Anti-igf-i receptor antibody

ABSTRACT

The present invention provides an anti-IGF-I receptor antibody that binds specifically to an IGF-I receptor of a vertebrate and has the proliferation-inducing activity of a vertebrate-derived cell, or a fragment thereof, or derivatives of these.

CROSS REFERENCE TO RELATED APPLICATIONS

This application is a Rule 53(b) Divisional of U.S. application Ser. No.17/595,959 filed Nov. 30, 2021, which is a National Stage ofInternational Application No. PCT/JP2018/020581 filed May 29, 2018,claiming priority based on Japanese Patent Application No. 2017-106529filed May 30, 2017, the above-noted applications incorporated herein byreference in their respective entireties.

INCORPORATION BY REFERENCE OF SEQUENCE LISTING

The content of the electronically submitted sequence listing, file name:Q284845_sequence listing as filed.xml; size: 89,126 bytes; and date ofcreation: Mar. 2, 2023, filed herewith, is incorporated herein byreference in its entirety.

FIELD

The present invention relates to an anti-IGF-I receptor antibody and,more specifically, to an anti-IGF-I receptor antibody which specificallybinds to an IGF-I receptor of a vertebrate.

BACKGROUND 1. IGF-I

IGF-I is an insulin-like growth factor secreted mainly from the liver,and affects an IGF-I receptor to thereby express a variety ofphysiological functions in various organs. Because of this, IGF-I isexpected to be used for the treatment of a variety of diseases. Sincethe amino acid sequence of IGF-I has a high similarity of about 40% tothat of proinsulin, IGF-I can bind to an insulin receptor and therebyexpress insulin-like effects (Non-Patent Literature 1). In addition,since the amino acid sequence of the IGF-I receptor has a highsimilarity of about 60% to that of an insulin receptor, these receptorscan form a heterodimer (Non-Patent Literature 1). Insulin can act on theinsulin receptor to thereby express a strong effect of lowering thelevel of blood glucose, and is therefore used as a hypoglycemic drug.

2. IGF-I Receptor

An IGF-I receptor is a transmembrane protein consisting of an alphachain and a beta chain, and has six extracellular domains (L1, CR, L2,Fn1, Fn2, and Fn3), a transmembrane domain, and an intracellular domain(Non-Patent Literature 2). The intracellular domain of the IGF-Ireceptor incorporates a tyrosine kinase. The extracellular domain is aCR (cysteine-rich) domain and participates in activation of theintracellular tyrosine kinase associated with conformational change ofthe IGF-I receptor, which occurs when IGF-I binds to the IGF-I receptor.The IGF-I receptor forms a homodimeric complex (homo form). IGF-Ibinding to the IGF-I receptor (homo form) triggers signaling viaactivation of the receptor kinase. The IGF-I receptor also forms aheterodimeric complex (hetero form) with the insulin receptor. Insulinor IGF-I binding to the IGF-I receptor (hetero form) triggers signalingvia activation of the receptor kinase (Non-Patent Literatures 3 and 4).

3. Physiological Effects of IGF-I

IGF-I has been shown to exhibit growth promoting effects, such asincreasing the body length and the body weight, and insulin-likemetabolic effects, such as glucose metabolism acceleration andhypoglycemic effects. It has been revealed that mecasermin, a humanrecombinant IGF-I, improves symptoms related to insulin receptorabnormality, such as hyperglycemia, hyperinsulinemia, acanthosisnigricans and hirsutism. IGF-I has also been shown to improve growthdisorder of dwarfism resistant to growth hormone (Non-Patent Literature5).

As its growth promoting effects, IGF-I is known to promote the DNAsynthetic capacity of human cartilage cells. It is also known thatadministration of IGF-I to a hypophysectomized rat increases its bodyweight and femur length (Non-Patent Literature 5).

4. Effect of IGF-I on Increasing Muscle Mass

Enhancement of cell proliferation activity with IGF-I requirescontinuous activation of the IGF-I receptor (Non-Patent Literature 6).An animal engineered to overexpress the IGF-I receptor exhibitsincreased muscle mass (Non-Patent Literature 7). Sustainedadministration of IGF-I/IGFBP3 to a patient with proximal femur fractureenhances her/his grip strength and improves her/his ability of standingfrom a seated position without assistance (Non-Patent Literature 8). Themuscle IGF-I levels of the elderly humans and mice are known to be lowerthan those of the young (Non-Patent Literatures 9 and 10). Overexpression of IGF-I specifically in muscle tissues of elderly miceimproved their muscle masses compared to wild-type mice (Non-PatentLiterature 11).

5. Precedent Products for Increasing Muscle Mass

Anamorelin, a ghrelin receptor agonist, increased lean body mass in aclinical trial for cachexia, which is a disuse muscle atrophy. However,it involves adverse effects such as inducing nausea and hyperglicemia(Non-Patent Literature 12).

Myostatin, a negative control factor of skeletal myogenesis, affectsactivin receptor II (ActRII) to thereby inhibit Akt/mTOR (Non-PatentLiteratures 13 to 15).

LY2495655, an anti-myostatin antibody, increases the muscle masses ofpatients who received total hip replacement arthroplasty and those ofelderly subjects (Non-Patent Literatures 16 and 17).

Bimagrumab, an anti-ActRII antibody, increases the muscle mass ofneuromuscular disease patients (Non-Patent Literature 18).

However, there is no drug so far which promotes formation of skeletalmuscles and can thereby be used for the treatment of a subject in needthereof.

6. Precedent Products for Promoting Growth

Human recombinant growth hormone (GH) formulation activates a GHreceptor and induces IGF-I secretion, thereby exhibiting growthpromoting effects. However, since the formulation requires once-dailyadministration via subcutaneous injection, it often causes poor drugcompliance (e.g., unintentional omission of medication) and results inreduction in growth effects (Non-Patent Literature 19). There is anongoing attempt to develop a long-acting GH formulation with improvedkinetics which is to be administered once every one or two weeks.

However, there is no drug so far which exhibits growth promoting effectsand can thereby be used for the treatment of a subject in need thereofwith improved drug compliance. In addition, the GH formulation has beenfound to exhibit reduced growth effects on patients of GH receptorabnormality with reduced sensitivity to activation of the GH receptor,or patients resistant to GH treatment (Non-Patent Literature 20).

IGF-I is the only therapeutic agent which has growth promoting effect ona patient having reduced sensitivity to the GH receptor activation,since it acts on any point downstream of the GH receptor. However, theIGF-I formulation is a parenteral solution to be administered twicedaily and therefore likely to cause poor drug compliance. In addition,it has been shown to cause hypoglycemia as an adverse effect (Non-PatentLiterature 21). There is no drug so far which has improved drugcompliance and reduced occurrence of hypoglycemia than IGF-I and can beused as an alternative therapeutic agent.

7. Hypoglycemic Effect of IGF-I

IGF-I is known to have hypoglycemic effect as an insulin-like effect.IGF-I enhances glucose uptake effect of rat muscle-derived cells(Non-Patent Literature 5). Administration of IGF-I also reduces theblood glucose level of rats (Non-Patent Literature 5).

It has been reported that the glucose lowering effect of IGF-I causehypoglycemia as a clinical adverse effect (Non-Patent Literature 21).Likewise, administration of IGF-I to a human subject causeshypoglycemia. Therefore, at the onset of IGF-I treatment, it isnecessary to keep controlling the dosage starting from a low dosage withobserving various clinical findings including the blood glucose levelafter administration (Non-Patent Literature 5).

IGF-I expresses hypoglycemic effect via promotion of Aktphosphorylation, which is a downstream signal of the IGF-I receptor. Anactive variant of Akt enhances glucose uptake by 3T3-L1 cells(Non-Patent Literature 22). On the other hand, an Akt2-deficient mouseexhibited elevated blood glucose level (Non-Patent Literature 23). AnAkt inhibitor inhibits insulin-induced glucose uptake by ratmuscle-derived cells (Non-Patent Literature 24). IGF-I is also known toactivate an insulin receptor which plays a role in hypoglycemic effect.These findings suggest that the hypoglycemic effect of IGF-I involvesoveractivation of Akt and activation of the insulin receptor.

8. Short Half-Life of IGF-I in Blood

IGF-I has a short half-life in blood, and therefore requires frequentadministrations when used in treatment. In fact, mecasermin, a humanrecombinant IGF-I, has a blood half-life of about 11 hours to about 16hours, and therefore needs to be administered once to twice daily in thetreatment of dwarfism (Non-Patent Literature 5).

About 70 to 80% of IGF-I is bound to IGFBP3 in blood, while a free formof IGF-I exhibits physiological effect. Binding of IGF-I to IGFBP3maintains its half-life in blood to a time period of from about 10 hoursto about 16 hours (Non-Patent Literature 1).

IPLEX, a combination drug of IGF-I with IGFBP3, exhibited a bloodhalf-life extended from that of IGF-I to a time period of about 21 hoursto about 26 hours, and thereby allowed for reduction of administrationfrequency to once daily (Non-Patent Literature 23). However, IPLEX wasalready withdrawn from the market.

There has been also an attempt to develop a PEGylated IGF-I withimproved IGF-I kinetics, but no drug has successfully been developed sofar and is currently available (Patent Literature 1).

9. Therapeutic Effects Expected to be Achieved Via IGF-I's Effects

IGF-I is known to affect various organs and exerts a wide variety ofphysiological functions (Non-Patent Literature 21).

IGF-I has been reported to have neuroprotective effect on the centralnervous system by protecting mitochondria and antioxidant effect viaactivation of the IGF-I receptor (Non-Patent Literatures 26 and 27).IGF-I promotes regeneration of injured neurites (Non-Patent Literature28).

IGF-I is a main factor of growth promotion (Non-Patent Literatures 29and 30). In fact, mecasermin, a human recombinant IGF-I, is clinicallyused as a drug for the treatment of dwarfism.

IGF-I is deemed to be effective in the treatment of hepatic cirrhosis,which evolves from liver damage or chronic liver disease and involveshepatic fibrosis. Administration of IGF-I improved hepatic fibrosis in amodel animal of hepatic cirrhosis (Non-Patent Literature 31).

IGF-I is also known to play a role in the development and functions ofkidney. IGF-I has protective effect against oxidative stress andapoptosis due to glucotoxicity in mesangial cells of kidney (Non-PatentLiterature 32). IGF-I is expected as a drug for the treatment ofnephropathy.

Examples of conditions expected to be improved via IGF-I administrationinclude: dwarfism, Laron syndrome, hepatic cirrhosis, hepatic fibrosis,aging, intrauterine growth restriction (IUGR), neurological disease,cerebral stroke, spinal cord injury, cardiovascular protection,diabetes, insulin resistant, metabolic syndrome, nephropathy,osteoporosis, cystic fibrosis, wound healing, myotonic dystrophy,AIDS-associated sarcopenia, HIV-associated fat redistribution syndrome,burn, Crohn's disease, Werner's syndrome, X-linked combinedimmunodeficiency disease, hearing loss, anorexia nervosa, andretinopathy of prematurity (Non-Patent Literature 21).

Thus, IGF-I is expected as a drug for the treatment of a variety ofdiseases because of its wide spectrum of physiological effects. However,problems such as its adverse hypoglycemic effect and its short half-liferequiring multiple administrations have prevented its clinicalapplications.

10. IGF-I Receptor Agonist Antibodies

In general, antibody formulations have long half-life, and proveeffective if administered once to twice a month. Although some IGF-Ireceptor agonist antibodies have been reported to be effective inactivating the receptor in vitro, no antibodies have been reported toexhibit agonistic activity against the IGF-I receptor in vivo(Non-Patent Literatures 33 to 37).

Specifically, antibodies 3B7 and 2D1 enhance cellular DNA synthesis invitro (Non-Patent Literature 34).

Antibodies 11A1, 11A4, 11A11, and 24-57 enhance tyrosine phosphorylationof IGF-I receptor in vitro (Non-Patent Literature 35).

Antibodies 16-13, 17-69, 24-57, 24-60, 24-31, and 26-3 are shown to beeffective in promoting cellular DNA synthesis and glucose uptake invitro, and have the potential to exhibit hypoglycemic effect (Non-PatentLiteratures 36 and 37).

However, no IGF-I receptor agonist antibody has been reported to exhibitcell proliferation effects in an in vitro experiment using primarycultured cells, inter alia, human myoblasts, let alone muscle-massincreasing effects in vivo.

11. IGF-I Receptor Antagonist Antibodies

There are attempts to use an antibody which binds to the IGF-I receptorfor the treatment of malignancies, based on its antagonist effect ofinhibiting binding of IGF-I to the IGF-I receptor. However, existingIGF-I receptor antagonist antibodies have various adverse effects suchas hyperglycemia in monotherapy (Non-Patent Literature 38), and exhibitincreased incidence of hyperglycemia when used in combination with otheranticancer agents (Non-Patent Literature 39). Accordingly, theirtherapeutic applications are expected to be limited.

LIST OF CITATIONS Patent Literature

-   [Patent Literature 1] Use of PEGylated Igf-I Variants for the    Treatment of Neuromuscular Disorders, JP2011-518778A    (WO2009/121759A) (2011)

Non-Patent Literature

-   [Non-Patent Literature 1] Ohlsson, C., et al., The role of    liver-derived insulin-like growth factor-I. Endocr Rev, 2009.    30(5): p. 494-535.-   [Non-Patent Literature 2] Kavran, J. M., et al., How IGF-I activates    its receptor. Elife, 2014. 3.-   [Non-Patent Literature 3] Bailyes, E. M., et al., Insulin    receptor/IGF-I receptor hybrids are widely distributed in mammalian    tissues: quantification of individual receptor species by selective    immunoprecipitation and immunoblotting. Biochem J, 1997. 327 (Pt    1): p. 209-15.-   [Non-Patent Literature 4] Pandini, G., et al., Insulin/insulin-like    growth factor I hybrid receptors have different biological    characteristics depending on the insulin receptor isoform involved.    J Biol Chem, 2002. 277(42): p. 39684-95.-   [Non-Patent Literature 5] OrphanPacific, IF. 2015.-   [Non-Patent Literature 6] Fukushima, T., et al.,    Phosphatidylinositol 3-kinase (PI3K) activity bound to insulin-like    growth factor-I (IGF-I) receptor, which is continuously sustained by    IGF-I stimulation, is required for IGF-I-induced cell proliferation.    J Biol Chem, 2012. 287(35): p. 29713-21.-   [Non-Patent Literature 7] Schiaffino, S. and C. Mammucari,    Regulation of skeletal muscle growth by the IGF-I-Akt/PKB pathway:    insights from genetic models. Skelet Muscle, 2011. 1(1): p. 4.-   [Non-Patent Literature 8] Boonen, S., et al., Musculoskeletal    effects of the recombinant human IGF-I/IGF binding protein-3 complex    in osteoporotic patients with proximal femoral fracture: a    double-blind, placebo-controlled pilot study. J Clin Endocrinol    Metab, 2002. 87(4): p. 1593-9.-   [Non-Patent Literature 9] Barton-Davis, E. R., et al., Viral    mediated expression of insulin-like growth factor I blocks the    aging-related loss of skeletal muscle function. Proc Natl Acad Sci    USA, 1998. 95(26): p. 15603-7.-   [Non-Patent Literature 10] Lamberts, S. W., A. W. van den Beld,    and A. J. van der Lely, The endocrinology of aging. Science, 1997.    278(5337): p. 419-24.-   [Non-Patent Literature 11] Musaro, A., et al., Localized IGF-I    transgene expression sustains hypertrophy and regeneration in    senescent skeletal muscle. Nat Genet, 2001. 27(2): p. 195-200.-   [Non-Patent Literature 12] Temel, J. S., et al., Anamorelin in    patients with non-small-cell lung cancer and cachexia (ROMANA 1 and    ROMANA 2): results from two randomized, double-blind, phase 3    trials. Lancet Oncol, 2016. 17(4): p. 519-31.-   [Non-Patent Literature 13] Glass, D. J., Signaling pathways    perturbing muscle mass. Curr Opin Clin Nutr Metab Care, 2010.    13(3): p. 225-9.-   [Non-Patent Literature 14] Lee, S. J. and A. C. McPherron,    Regulation of myostatin activity and muscle growth. Proc Natl Acad    Sci USA, 2001. 98(16): p. 93 06-11.-   [Non-Patent Literature 15] Amirouche, A., et al., Down-regulation of    Akt/mammalian target of rapamycin signaling pathway in response to    myostatin overexpression in skeletal muscle. Endocrinology, 2009.    150(1): p. 286-94.-   [Non-Patent Literature 16] Woodhouse, L., et al., A Phase 2    Randomized Study Investigating the Efficacy and Safety of Myostatin    Antibody LY2495655 versus Placebo in Patients Undergoing Elective    Total Hip Arthroplasty. J Frailty Aging, 2016. 5(1): p. 62-70.-   [Non-Patent Literature 17] Becker, C., et al., Myostatin antibody    (LY2495655) in older weak fallers: a proof-of-concept, randomized,    phase 2 trial. Lancet Diabetes Endocrinol, 2015. 3(12): p. 948-57.-   [Non-Patent Literature 18] Amato, A. A., et al., Treatment of    sporadic inclusion body myositis with bimagrumab. Neurology, 2014.    83(24): p. 2239-46.-   [Non-Patent Literature 19] Cutfield, W. S., et al., Non-compliance    with growth hormone treatment in children is common and impairs    linear growth. PLos One., 2011.6(1):e16223-   [Non-Patent Literature 20] Bang, P., et al., Identification and    management of poor response to growth-promoting therapy in children    with short stature. Clin Endocrinol (Oxf)., 2012.77(2): p. 169-181.-   [Non-Patent Literature 21] Puche, J. E. and I. Castilla-Cortazar,    Human conditions of insulin-like growth factor-I (IGF-I) deficiency.    J Transl Med, 2012. 10: p. 224.-   [Non-Patent Literature 22] Kohn, A. D., et al., Expression of a    constitutively active Akt Ser/Thr kinase in 3T3-L1 adipocytes    stimulates glucose uptake and glucose transporter 4 translocation. J    Biol Chem, 1996. 271(49): p. 31372-8.-   [Non-Patent Literature 23] Cho, H., et al., Insulin resistance and a    diabetes mellitus-like syndrome in mice lacking the protein kinase    Akt2 (PKB beta). Science, 2001. 292(5522): p. 1728-31.-   [Non-Patent Literature 24] Green, C. J., et al., Use of Akt    inhibitor and a drug-resistant mutant validates a critical role for    protein kinase B/Akt in the insulin-dependent regulation of glucose    and system A amino acid uptake. J Biol Chem, 2008. 283(41): p.    27653-67.-   [Non-Patent Literature 25] Submission for marketing application to    FDA, APPLICATION NUMBER, 21-884-   [Non-Patent Literature 26] Garcia-Fernandez, M., et al., Low doses    of insulin-like growth factor I improve insulin resistance, lipid    metabolism, and oxidative damage in aging rats. Endocrinology, 2008.    149(5): p. 2433-42.-   [Non-Patent Literature 27] Puche, J. E., et al., Low doses of    insulin-like growth factor-I induce mitochondrial protection in    aging rats. Endocrinology, 2008. 149(5): p. 2620-7.-   [Non-Patent Literature 28] Joseph D'Ercole, A. and P. Ye, Expanding    the mind: insulin-like growth factor I and brain development.    Endocrinology, 2008. 149(12): p. 5958-62.-   [Non-Patent Literature 29] Abuzzahab, M. J., et al., IGF-I receptor    mutations resulting in intrauterine and postnatal growth    retardation. N Engl J Med, 2003. 349(23): p. 2211-22.-   [Non-Patent Literature 30] Woods, K. A., et al., Intrauterine growth    retardation and postnatal growth failure associated with deletion of    the insulin-like growth factor I gene. N Engl J Med, 1996.    335(18): p. 1363-7.-   [Non-Patent Literature 31] Perez, R., et al., Mitochondrial    protection by low doses of insulin-like growth factor-I in    experimental cirrhosis. World J Gastroenterol, 2008. 14(17): p.    2731-9.-   [Non-Patent Literature 32] Kang, B. P., et al., IGF-I inhibits the    mitochondrial apoptosis program in mesangial cells exposed to high    glucose. Am J Physiol Renal Physiol, 2003. 285(5): p. F1013-24.-   [Non-Patent Literature 33] Bhaskar, V., et al., A fully human,    allosteric monoclonal antibody that activates the insulin receptor    and improves glycemic control. Diabetes, 2012. 61(5): p. 1263-71.-   [Non-Patent Literature 34] Xiong, L., et al., Growth-stimulatory    monoclonal antibodies against human insulin-like growth factor I    receptor. Proc Natl Acad Sci USA, 1992. 89(12): p. 5356-60.-   [Non-Patent Literature 35] Runnels, H. A., et al., Human monoclonal    antibodies to the insulin-like growth factor 1 receptor inhibit    receptor activation and tumor growth in preclinical studies. Adv    Ther, 2010. 27(7): p. 458-75.-   [Non-Patent Literature 36] Soos, M. A., et al., A panel of    monoclonal antibodies for the type I insulin-like growth factor    receptor. Epitope mapping, effects on ligand binding, and biological    activity. J Biol Chem, 1992. 267(18): p. 12955-63.-   [Non-Patent Literature 37] Kato, H., et al., Role of tyrosine kinase    activity in signal transduction by the insulin-like growth factor-I    (IGF-I) receptor. Characterization of kinase-deficient IGF-I    receptors and the action of an IGF-I-mimetic antibody (alpha IR-3).    J Biol Chem, 1993. 268(4): p. 2655-61.-   [Non-Patent Literature 38] Atzori, F., et al., A Phase I    Pharmacokinetic and Pharmacodynamic Study of Dalotuzumab (MK-0646),    an Anti-Insulin-like Growth Factor-1 Receptor Monoclonal Antibody,    in Patients with Advanced Solid Tumors. Clin Cancer Res.,    2011.17(19): p. 6304-12.-   [Non-Patent Literature 39] de Bono J. S., et al., Phase II    randomized study of figitumumab plus docetaxel and docetaxel alone    with crossover for metastatic castration-resistant prostate cancer.    Clin Cancer Res., 2014.20(7): p. 1925-34.

SUMMARY Problem to be Solved by the Invention

An objective of the present invention is to provide an anti-IGF-Ireceptor antibody or its fragment or a derivative thereof whichspecifically binds to an IGF-I receptor of a vertebrate. Anotherobjective of the present invention is to provide an antibody whichincreases the muscle mass or the thickness of growth plate cartilage viathe IGF-I receptor while not reducing the blood glucose level.

Means to Solve the Problem

The present invention relates to the following:

Aspect [1] An anti-IGF-I receptor antibody or its fragment or aderivative thereof which specifically binds to an IGF-I receptor of avertebrate, and exhibits an activity to induce growth ofvertebrate-derived cells.Aspect [2] The anti-IGF-I receptor antibody or its fragment or aderivative thereof according to Aspect [1], wherein the activity of theantibody, fragment, or derivative to induce growth of vertebrate-derivedcells is equal to or higher than the corresponding activity of awild-type IGF-I.Aspect [3] The anti-IGF-I receptor antibody or its fragment or aderivative thereof according to Aspect [1] or Aspect [2], wherein theEC₅₀ value of the antibody, fragment, or derivative for inducing growthof vertebrate-derived cells in vitro is 1/20 or less of thecorresponding value of a wild-type IGF-I.Aspect [4] The anti-IGF-I receptor antibody or its fragment or aderivative thereof according to any one of Aspect [1] to Aspect [3],wherein when the antibody, fragment, or derivative is contacted withcultured vertebrate-derived cells, the duration of activity of theantibody, fragment, or derivative to induce growth of the culsturedcells relative to the duration of contact is improved than a wild-typeIGF-I.Aspect [5] The anti-IGF-I receptor antibody or its fragment or aderivative thereof according to any one of Aspect [2] to Aspect [4],wherein the wild-type IGF-I is a human IGF-I having an amino acidsequence defined in SEQ ID NO:1.Aspect [6] The anti-IGF-I receptor antibody or its fragment or aderivative thereof according to any one of Aspect [1] to Aspect [5],wherein the EC₅₀ value of the antibody, fragment, or derivative forinducing growth of vertebrate-derived cells in vitro is 0.1 nmol/L orlower.Aspect [7] The anti-IGF-I receptor antibody or its fragment or aderivative thereof according to any one of Aspect [1] to Aspect [6],which exhibits an activity to induce an increase in the muscle massand/or the body length of a vertebrate when parenterally administered tothe vertebrate.Aspect [8] The anti-IGF-I receptor antibody or its fragment or aderivative thereof according to any one of Aspect [1] to Aspect [7],which is administered to a vertebrate at a frequency of once a week orless.Aspect [9] The anti-IGF-I receptor antibody or its fragment or aderivative thereof according to any one of Aspect [1] to Aspect [8],wherein the vertebrate is a human; a non-human animal including a guineapig, a monkey, a rabbit, a cow, a pig, a horse, a sheep, a dog, or afowl; or a non-human animal engineered to express a human IGF-Ireceptor.Aspect [10] The anti-IGF-I receptor antibody or its fragment or aderivative thereof according to any one of Aspect [1] to Aspect [9],which does not induce glucose uptake by differentiated muscle cells whenadministered at a dosage sufficient to induce growth ofvertebrate-derived cells.Aspect [11] The anti-IGF-I receptor antibody or its fragment or aderivative thereof according to Aspect [10], which does not induceglucose uptake by differentiated muscle cells when administered at adosage of 100 times or more of the EC₅₀ value for inducing growth ofvertebrate-derived cells in vitro.Aspect [12] The anti-IGF-I receptor antibody or its fragment or aderivative thereof according to Aspect [10] or Aspect [11], wherein thevertebrate-derived cells are myoblasts derived from a human or anon-human mammal.Aspect [13] The anti-IGF-I receptor antibody or its fragment or aderivative thereof according to any one of Aspect [7] to Aspect [12],which does not lower the blood glucose level of a vertebrate whenparenterally administered to the vertebrate at a dosage sufficient toinduce an increase in the muscle mass and/or the body length of thevertebrate.Aspect [14] The anti-IGF-I receptor antibody or its fragment or aderivative thereof according to Aspect [13], which does not change theblood glucose level of a vertebrate when parenterally administered tothe vertebrate at a dosage of 10 times or more of an effective dosagesufficient to induce an increase in the muscle mass and/or the bodylength of the vertebrate.Aspect [15] An anti-IGF-I receptor antibody or its fragment or aderivative thereof, according to any one of Aspect [1] to Aspect [14],which binds to a CR domain of an IGF-I receptor.Aspect [16] An anti-IGF-I receptor antibody or its fragment or aderivative thereof, which binds to a CR domain of an IGF-I receptor, andinhibits binding of IGF-I or IGF-II to an IGF-I receptor.Aspect [17] The anti-IGF-I receptor antibody or its fragment or aderivative thereof according to Aspect [16], which binds to an epitopecontaining ProSerGlyPheIleArgAsnX₁×₂GlnSerMet (where X₁ represents Glyor Ser and X₂ represents Ser or Thr) (SEQ ID NO: 31), or a part in thevicinity thereof, in the sequence of the CR domain of the IGF-Ireceptor.Aspect [18] The anti-IGF-I receptor antibody or its fragment or aderivative thereof according to Aspect [17], which binds to an epitopecontaining ProSerGlyPheIleArgAsnGlySerGlnSerMet (SEQ ID NO: 32), or apart in the vicinity thereof, in the sequence of the CR domain of theIGF-I receptor.Aspect [19] The anti-IGF-I receptor antibody or its fragment or aderivative thereof according to any one of Aspect [1] to Aspect [18],which has a cross-reactivity with an IGF-I receptor of a human or anon-human animal including a guinea pig, a monkey, a rabbit, a cow, apig, a horse, a sheep, a dog, or a fowl.Aspect [20] The anti-IGF-I receptor antibody or its fragment or aderivative thereof according to any one of Aspect [1] to Aspect [19],which causes an antigen-antibody reaction with an affinity intensity ata equilibrium dissociation constant (KD) of 1×10⁻⁸M or less.Aspect [21] The anti-IGF-I receptor antibody or its fragment or aderivative thereof according to any one of Aspect [16] to Aspect [20],which has at least one of the features of:1) exhibiting an activity to induce growth of vertebrate-derived cells;2) exhibiting an activity to induce an increase in the muscle massand/or the body length of a vertebrate when parenterally administered tothe vertebrate;3) not inducing glucose uptake by differentiated muscle cells whenadministered at a dosage sufficient to induce growth ofvertebrate-derived cells; and4) not changing the blood glucose level of a vertebrate whenparenterally administered to the vertebrate at a dosage sufficient toinduce an increase in the muscle mass and/or the body length of thevertebrate.Aspect [22] The anti-IGF-I receptor antibody or its fragment or aderivative thereof according to any one of Aspect [16] to Aspect [21],which has at least one of the features of:1) inhibiting growth of vertebrate-derived cells induced by IGF-I;2) inhibiting IGF-I-induced cell proliferation in a vertebrate sufferinga cell proliferative disease when parenterally administered to thevertebrate;3) not affecting glucose uptake by differentiated muscle cells at adosage sufficient to inhibit growth of vertebrate-derived cells inducedby IGF-I; and4) not changing the blood glucose level of a vertebrate suffering a cellproliferative disease when parenterally administered to the vertebrateat a dosage sufficient to inhibit IGF-I-induced cell proliferation inthe vertebrate.Aspect [23] The anti-IGF-I receptor antibody or its fragment or aderivative thereof according to any one of Aspect [1] to Aspect [22],which is a Fab, scFv, diabody or bispecific antibody, or a derivativethereof.Aspect [24] The anti-IGF-I receptor antibody or its fragment or aderivative thereof according to any one of Aspect [1] to Aspect [23],consisting of an amino acid sequence comprising:

as a heavy chain variable region CDR-1 (CDR-H1) sequence, an amino acidsequence defined in SEQ ID NO:3 or an amino acid sequence derived fromSEQ ID NO:3 via substitution, deletion or insertion of any one aminoacid residue;

as a heavy chain variable region CDR-2 (CDR-H2) sequence, an amino acidsequence defined in SEQ ID NO:4 or an amino acid sequence derived fromSEQ ID NO:4 via substitution, deletion or insertion of any one or twoamino acid residues;

as a heavy chain variable region CDR-3 (CDR-H3) sequence, an amino acidsequence defined in SEQ ID NO:5 or an amino acid sequence derived fromSEQ ID NO:5 via substitution, deletion or insertion of any one or twoamino acid residues;

as a light chain variable region CDR-1 (CDR-L1) sequence, an amino acidsequence defined in SEQ ID NO:6 or an amino acid sequence derived fromSEQ ID NO:6 via substitution, deletion or insertion of any one or twoamino acid residues;

as a light chain variable region CDR-2 (CDR-L2) sequence, an amino acidsequence defined in SEQ ID NO:7 or an amino acid sequence derived fromSEQ ID NO:7 via substitution, deletion or insertion of any one aminoacid residue; and

as a light chain variable region CDR-3 (CDR-L3) sequence, an amino acidsequence defined in SEQ ID NO:8 or an amino acid sequence derived fromSEQ ID NO:8 via substitution, deletion or insertion of any one or twoamino acid residues.

Aspect [25] The anti-IGF-I receptor antibody or its fragment or aderivative thereof according to Aspect [24 further comprises a frameworksequence of immunoglobulin.Aspect [26] The anti-IGF-I receptor antibody or its fragment or aderivative thereof according to Aspect [25], wherein the frameworksequence of immunoglobulin is a framework sequence of each class ofimmunoglobulin from a human or a non-human animal including a guineapig, a monkey, a rabbit, a cow, a pig, a horse, a sheep, a dog, a fowl,a mouse, or a rat.Aspect [27] The anti-IGF-I receptor antibody or its fragment or aderivative thereof according to any one of Aspect [1] to Aspect [26],which consists of an amino acid sequence comprising:

as a heavy chain variable region, an amino acid sequence defined in SEQID NO:9 or an amino acid sequence having a similarity of 90% or more toSEQ ID NO:9; and

as a light chain variable region, an amino acid sequence defined in SEQID NO:10 or an amino acid sequence having a similarity of 90% or more toSEQ ID NO:10.

Aspect [28] The anti-IGF-I receptor antibody or its fragment or aderivative thereof according to any one of Aspect [1] to Aspect [27]further comprising a constant region of each class of immunoglobulin ahuman or a non-human animal including a guinea pig, a monkey, a rabbit,a cow, a pig, a horse, a sheep, a dog, a fowl, a mouse, or a rat.Aspect [29] A nucleic acid molecule consisting of a polynucleotidesequence encoding an anti-IGF-I receptor antibody or its fragment or aderivative thereof according to any one of

Aspect [1] to Aspect [28].

Aspect [30] A cloning vector or expression vector comprising at leastone nucleic acid molecule according to Aspect [29].Aspect [31] A recombinant cell derived from a host cell via transfectionof a vector according to Aspect [30].Aspect [32] A process of producing an anti-IGF-I receptor antibody orits fragment or a derivative thereof according to any one of Aspect [1]to Aspect [28], comprising:

culturing a recombinant cell according to Aspect [31]; and

purifying the anti-IGF-I receptor antibody, fragment, or derivativeproduced from the recombinant cell.

Aspect [33] A pharmaceutical composition comprising an anti-IGF-Ireceptor antibody or its fragment or a derivative thereof according toany one of Aspect [1] to Aspect [28], a nucleic acid molecule accordingto Aspect [29], a vector according to Aspect [30], or a recombinant cellaccording to Aspect [31].Aspect [34] The pharmaceutical composition according to Aspect [33],further comprising an additional active ingredient other than theanti-IGF-I receptor antibody, fragment, or derivative according to anyone of Aspect [1] to Aspect [28], the nucleic acid molecule according toAspect [29], the vector according to Aspect [30], or the recombinantcell according to Aspect [31].Aspect [35] The pharmaceutical composition according to Aspect [34],wherein the active ingredient is one or more selected from a growthhormone or an analog thereof, insulin or an analog thereof, IGF-II or ananalog thereof, an anti-myostatin antibody, a myostatin antagonist, ananti-activin type IIB receptor antibody, an activin type IIB receptorantagonist, a soluble activin type IIB receptor or an analog thereof,ghrelin or an analog thereof, follistatin or an analog thereof, a beta-2agonist, and a selective androgen receptor modulator.Aspect [36] The pharmaceutical composition according to Aspect [34] orAspect [35], wherein the active ingredient comprises an ingredientselected from the group consisting of: corticosteroid, antiemetic,ondansetron hydrochloride, granisetron hydrochloride, metoclopramide,domperidone, haloperidol, cyclizine, lorazepam, prochlorperazine,dexamethasone, levomepromazine, tropisetron, cancer vaccine, GM-CSFinhibitor, GM-CSF DNA vaccine, cell-based vaccine, dendritic cellvaccine, recombinant virus vaccine, heat shock protein (HSP) vaccine,homologous tumor vaccine, autologous tumor vaccine, analgesic,ibuprofen, naproxen, choline magnesium trisalicylate, oxycodonehydrochloride, anti-angiogenic, antithrombotic, anti-PD-1 antibody,nivolumab, pembrolizumab, anti-PD-L1 antibody, atezolizumab, anti-CTLA4antibody, ipilimumab, anti-CD20 antibody, rituximab, anti-HER2 antibody,trastuzumab, anti-CCR4 antibody, mogamulizumab, anti-VEGFantibody,bevacizumab, anti-VEGF receptor antibody, soluble VEGF receptorfragment, anti-TWEAK antibody, anti-TWEAK receptor antibody, solubleTWEAK receptor fragment, AMG 706, AMG 386, antiproliferative, farnesylprotein transferase inhibitor, alpha v beta 3 inhibitor, alpha v beta 5inhibitor, p53 inhibitor, Kit receptor inhibitor, ret receptorinhibitor, PDGFR inhibitor, growth hormone secretion inhibitor,angiopoietin inhibitor, tumor-infiltrating macrophage inhibitor, c-fmsinhibitor, anti-c-fms antibody, CSF-1 inhibitor, anti-CSF-1 antibody,soluble c-fms fragment, pegvisomant, gemcitabine, panitumumab,irinotecan, and SN-38.Aspect [37] A medical drug for use in the treatment or prevention of acondition associated with IGF-I or IGF-II, comprising comprising ananti-IGF-I receptor antibody or its fragment or a derivative thereofaccording to any one of Aspect [1] to Aspect [28], a nucleic acidmolecule according to Aspect [29], a vector according to Aspect [30], ora recombinant cell according to Aspect [31].Aspect [38] The medical drug according to Aspect [37], wherein thecondition associated with IGF-I is selected from: disuse muscle atrophy,dwarfism, diabetic nephropathy, chronic renal failure, Laron syndrome,hepatic cirrhosis, hepatic fibrosis, aging, intrauterine growthrestriction (IUGR), neurological disease, cerebral stroke, spinal cordinjury, cardiovascular protection, diabetes, insulin resistant,metabolic syndrome, osteoporosis, cystic fibrosis, wound healing,myotonic dystrophy, AIDS-associated sarcopenia, HIV-associated fatredistribution syndrome, burn, Crohn's disease, Werner's syndrome,X-linked combined immunodeficiency disease, hearing loss, anorexianervosa and retinopathy of prematurity, Turner's syndrome, Prader-Willisyndrome, Silver-Russell syndrome, idiopathic short stature, obesity,multiple sclerosis, fibromyalgia, ulcerous colitis, low muscle mass,myocardial ischemia and decreased bone density.Aspect [39] The medical drug according to Aspect [37] or Aspect [38],which is parenterally administered.Aspect [40] The medical drug according to any one of Aspect [37] toAspect [39], which is a veterinary drug to be administered to anon-human animal.Aspect [41] The medical drug according to Aspect [40], wherein theveterinary drug is administered for the purpose of, increasing musclemass and/or body length, promoting growth, increasing milk production,promoting reproduction, or preventing aging.Aspect [42] The medical drug according to Aspect [40] or Aspect [41],wherein the non-human animal is a guinea pig, a monkey, a rabbit, a cow,a pig, a horse, a sheep, a dog, or a fowl.Aspect [43] The medical drug according to any one of Aspect [37] toAspect [42], for the treatment or prevention of a disease caused by aneffect of IGF-I or IGF-II on an IGF-I receptor.Aspect [44] The medical drug according to Aspect [43], wherein thedisease caused by an effect of IGF-I or IGF-II on an IGF-I receptor isselected from the group consisting of: liver cancer, neuroblastoma,striated muscle sarcoma, bone cancer, childhood cancer, acromegalia,ovary cancer, pancreas cancer, benignant prostatic hypertrophy, breastcancer, prostate cancer, bone cancer, lung cancer, colorectal cancer,cervix cancer, synovial sarcoma, urinary bladder cancer, stomach cancer,Wilms' tumor, diarrhea associated with metastatic carcinoid andvasoactive intestinal peptide secreting tumor, vipoma, Verner-Morrisonsyndrome, Beckwith-Wiedemann syndrome, kidney cancer, renal cell cancer,transitional cell cancer, Ewing's sarcoma, leukemia, acute ymphoblasticleukemia, brain tumor, glioblastoma, non-glioblastomatic brain tumor,meningioma, pituitary adenoma, vestibular schwannoma, primitiveneuroectodermal tumor, medulloblastoma, astrocytoma, oligodendroglioma,ependymoma, choroid plexus papilloma, gigantism, psoriasis,atherosclerosis, vascular smooth muscle restenosis, inappropriatemicrovascular growth, diabetic retinopathy, Graves' disease, multiplesclerosis, systemic lupus erythematosus, chronic thyroiditis, myastheniagravis, autoimmune thyroiditis and Behcet's disease.Aspect [45] A method of culturing vertebrate-derived cells in vitro,comprising contacting the vertebrate-derived cells with an anti-IGF-Ireceptor antibody or its fragment or a derivative thereof according toany one of Aspect [1] to Aspect [28], a nucleic acid molecule accordingto Aspect [29], a vector according to Aspect [30], and a recombinantcell according to Aspect [31] when culturing the cells.Aspect [46] The method according to Aspect [45], wherein said contactingis carried out for the purpose of promoting growth or inducingdifferentiation of the vertebrate-derived cells.Aspect [47] The method according to Aspect [45] or Aspect [46], whereinthe anti-IGF-I receptor antibody, fragment, or derivative is adsorbedby, or immobilized to, a solid phase.Aspect [48] A transgenic animal comprising an IGF-I receptor gene whichhas been mutated in a CR domain thereof via gene engineering such thatthe CR domain includes an amino acid sequence represented byProSerGlyPheIleArgAsnGlySerGlnSerMet (SEQ ID NO: 32).Aspect [49] A transgenic animal into which a heterologous IGF-I receptorgene has been transfected, wherein the amino acid sequence encoded bythe heterologous IGF-I receptor gene differs from the amino acidsequence encoded by the animal's inherent IGF-I receptor gene in aminoacid residue(s) X₁ and/or X₂ of a sequence represented byProSerGlyPheIleArgAsnX₁×₂GlnSerMet (SEQ ID NO: 31) in a CR domain.

Effect of the Invention

The anti-IGF-I receptor antibody or its fragment or a derivative thereofaccording to the present invention has an effect of specifically bindingto an IGF-I receptor of a vertebrate.

BRIEF EXPLANATION OF THE DRAWINGS

FIG. 1 illustrates aligned amino acid sequences of CR domains of themouse (residues 121-360 of SEQ ID NO: 15), rat (residues 121-360 of SEQID NO: 14), human (residues 121-359 of SEQ ID NO: 2), guinea pig(residues 121-359 of SEQ ID NO: 11) and rabbit (residues 121-359 of SEQID NO: 13) IGF-I receptors, in which the amino acid sequences areindicated using the one letter code;

FIG. 2 is a graph indicating the results of ELISA using variants of apresumptive epitope of IGF11-16;

FIG. 3 is a graph indicating the growth activity of human myoblastsafter removal of IGF11-16 and IGF-I;

FIG. 4 is a graph indicating the glucose uptake effect by humandifferentiated muscle cells after addition of IGF-I and IGF11-16;

FIG. 5 is a graph indicating the weights of extensor digitorum longusmuscles of guinea pigs which received sustained administration of IGF-Iusing an osmotic pump for two weeks or single-dose subcutaneous orintravenous administration of IGF11-16 two weeks ago;

FIG. 6 is a graph indicating the time course of the blood glucose levelof guinea pigs under fasting conditions after single-dose subcutaneousadministration of IGF-I;

FIG. 7 is a graph indicating the time course of the blood glucose levelof guinea pigs under fasting conditions after single-dose subcutaneousadministration of IGF11-16;

FIG. 8 is a graph indicating the time course of the blood glucose levelof guinea pigs under fasting conditions after single-dose intravenousadministration of IGF11-16;

FIG. 9 is a graph indicating the effects of IGF11-16 in increasing thethickness of growth plate cartilage of hypophysectomized guinea pigs(HPX);

FIG. 10 is a graph indicating the effects of IGF11-16 in increasing thelength of tibia in hypophysectomized guinea pigs (HPX);

FIG. 11 is a graph indicating the blood kinetics of IGF-I in guinea pigsunder fasting conditions after single-dose subcutaneous administration;and

FIG. 12 is a graph indicating the blood kinetics of IGF11-16 in guineapigs under fasting conditions after single-dose subcutaneousadministration.

MODES FOR CARRYING OUT THE INVENTION

In the following description, the present invention will be explainedwith reference to specific embodiments, although the present inventionshould not be limited to these embodiments in any way. All the documentscited in the present specification, including patent publications,unexamined application publications, and non-patent literatures, arehereby incorporated by reference in their entirety for all purposes.

[IGF]

IGF refers to an insulin-like growth factor, which may be either IGF-Ior IGF-II. Both IGF-I and IGF-II are biological ligands having agonistactivities which bind to an IGF-I receptor (insulin-like growth factor-Ireceptor) and transduce signals such as cell division and metabolisminto the cell. IGF-I and IGF-II are also known to have cross-bindingactivity to an insulin receptor (INSR), which is structurally similar tothe IGF-I receptor. The present specification will mainly discuss IGF-I,since its properties such as physiological functions are known more thanthose of IGF-II. However, in the context of discussion about variouseffects and diseases mediated via binding of a ligand to the IGF-Ireceptor, both IGF-I and IGF-II may collectively be mentioned.

IGF-I, also referred to as somatomedin C, is a single polypeptidehormone consisting of 70 amino acids. The sequence of human IGF-I isavailable, e.g., on the EMBL-EBI with UniProtKB accession number P50919.The amino acid sequence of mature IGF-I is shown in SEQ ID NO:1 of thesequence listing attached hereto. This 70 amino acid sequence isconserved in many species. In the present invention, the term “IGF-I”without any limitation means an IGF-I protein having such hormoneactivity, unless specified otherwise.

IGF-I is produced by a variety of cells in the living body, includingliver cells, and exists in blood and other body fluids. Therefore,wild-type IGF-I can be obtained via purification from body fluid of ananimal or from a primary cultured cell or a cultured cell line derivedfrom an animal. Since a growth hormone induces IGF-I production bycells, IGF-I can also be purified from body fluid of an animal to whicha growth hormone has been administered, or from a primary culturedanimal cell or an animal cell line incubated in the presence of a growthhormone. As a different method, IGF-I can also be obtained from arecombinant cell prepared by transfection of an expression vectorcarrying a nucleic acid molecule encoding an amino acid sequence ofIGF-I into a host such as a prokaryotic organism (e.g., E. coli) or aeukaryotic cell including a yeast, an insect cell, or a culturedmammal-derived cell, or from a transgenic animal or a transgenic plantinto which an IGF-I gene has been transfected. Human IGF-I is alsoavailable as a research reagent (Enzo Life Sciences, catalog:ADI-908-059-0100, Abnova, catalog: P3452, etc.) or as a pharmaceuticalproduct (SOMAZON mecasermin, INCRELEX, etc.). The in vivo and in vitroactivities of IGF-I for use can be evaluated as specific activitiesrelative to an IGF-I standard substance under NIBSC code: 91/554, whoseactivity corresponds to one international unit/microgram. The standardsubstance is available from World Health Organization's NationalInstitute for Biological Standards and Control (NIBSC). In the contextof the present invention, IGF-I is considered as having a specificactivity equivalent to the IGF-I of NIBSC code: 91/554.

[IGF-I Receptor]

The term “IGF-I receptor” refers to an insulin-like growth factor-Ireceptor. The term “IGF-I receptor” used herein means an IGF-I receptorprotein, unless specified otherwise. The IGF-I receptor is a proteinformed with two subunits, each consisting of an alpha chain and a betachain. The amino acid sequence of a human IGF-I receptor is indicated inSEQ ID NO:2, of which a subsequence consisting of the 31^(st) to735^(th) amino acid residues represents the alpha chain, while asubsequence starting from the 740^(th) amino acid residue represents thebeta chain. The alpha chain of the IGF-I receptor has a portion to whichIGF-I binds, while the beta chain has a transmembrane structure andexhibits a function to transmit signals into the cell. The alpha chainof the IGF-I receptor can be divided into L1, CR, L2, FnIII-1, andFnIII-2a/ID/FnIII-2b domains. According to the amino acid sequence ofthe human IGF-I receptor defined in SEQ ID NO:2, the 31^(st) to 179^(th)residues correspond to the L1 domain, the 180^(th) to 328^(th) residuescorrespond to the CR domain, the 329^(th) to 491^(st) residuescorrespond to the L2 domain, the 492^(nd) to 607^(th) residuescorrespond to the FnIII-1 domain, and the 608^(th) to 735^(th) residuescorrespond to the FnIII-2a/ID/FnIII-2b domain. Among them, the CR(cysteine-rich) domain is involved in the activation of an intracellulartyrosine kinase in the beta chain, which is associated with aconformational change of the IGF-I receptor occurring when IGF-I bindsto the receptor. The amino acid sequence of human IGF-I receptor isavailable, e.g., on EMBL-EBI with UniProtKB-accession number P08069, andis also indicated in the sequence listing as SEQ ID NO:2.

The IGF-I receptor is known to be expressed in a wide range of tissuesand cells in the living body, and receives various stimuli via IGF-I,such as induction of cell proliferation and activation of intracellularsignals. In particular, effects of IGF-I on myoblasts via the IGF-Ireceptor can be evaluated using cell proliferation activities asindicators. For this reason, myoblasts are useful in analyzing theeffects of antibodies binding to the IGF-I receptor. Cells expressing anIGF-I receptor derived from human or any other vertebrate can beprepared artificially, by transfection of an expression vector carryinga nucleic acid molecule encoding the amino acid sequence of an IGF-Ireceptor derived from human or any other vertebrate into a eukaryotichost cell, such as a cultured insect cell or a mammal-derived cell, toprepare a recombinant cell expressing the IGF-I receptor encoded by thetransfected nucleic acid on its cell membrane. The resultant cellexpressing the IGF-I receptor can be used for analysis of the bindingability and intracellular signal transmissibility of antibodies.

[Anti-IGF-I Receptor Antibody]

An antibody is a glycoprotein containing at least two heavy (H) chainsand two light (L) chains coupled together via disulfide bindings. Eachheavy chain has a heavy chain variable region (abbreviated as VH) and aheavy chain constant region. The heavy chain constant region containsthree domains, i.e., CH1, CH2, and CH3. Each light chain contains alight chain variable region (abbreviated as VL) and a light chainconstant region. A light chain constant region has one domain, i.e., CL.There are two types of light chain constant regions, i.e., λ (lambda)chain and κ (kappa) chain. Heavy chain constant regions are classifiedinto γ (gamma) chain, μ (mu) chain, α (alpha) chain, δ (delta) chain andε (epsilon) chain, and different types of heavy chain constant regionsresult in different isotypes of antibodies, i.e., IgG, IgM, IgA, IgD,and IgE, respectively. Each of the VH and VL regions is also dividedinto four relatively conserved regions (FR-1, FR-2, FR-3, and FR-4),collectively referred to as framework regions (FR), and three highlyvariable regions (CDR-1, CDR-2, and CDR-3), collectively referred to ascomplementarity determining regions (CDR). The VH region includes thethree CDRs and the four FRs arranged in the order of FR-1, CDR-1(CDR-H1), FR-2, CDR-2 (CDR-H2), FR-3, CDR-3 (CDR-H3), and FR-4 from theamino terminal to the carboxyl terminal. The VL includes the three CDRsand the four FRs arranged in the order of FR-1, CDR-1 (CDR-L1), FR-2,CDR-2 (CDR-L2), FR-3, CDR-3 (CDR-L3), and FR-4 from the amino terminalto the carboxyl terminal. The variable region of each of the heavy chainand the light chain includes a binding domain, which interacts with anantigen.

The antibody according to the present invention may be a fragment and/orderivative of an antibody. Examples of antibody fragments includeF(ab′)2, Fab, and Fv. Examples of antibody derivatives include:antibodies to which an amino acid mutation has been introduced in itsconstant region; antibodies in which the domain arrangement of theconstant regions has been modified; antibodies having two or more Fc'sper molecule; antibodies consisting only of a heavy chain or only of alight chain; antibodies with modified glycosylation; bispecificantibodies; conjugates of antibodies or antibody fragments withcompounds or proteins other than antibodies; antibody enzymes;nanobodies; tandem scFv's; bispecific tandem scFv's; diabodies; andVHHs. The term “antibody” used herein encompasses such fragments and/orderivatives of antibodies, unless otherwise specified.

The term “monoclonal antibody” conventionally means antibody moleculesobtained from a clone derived from a single antibody-producing cell,i.e., a single variety of antibody molecules having a combination of VHand VL with specific amino acid sequences. A monoclonal antibody canalso be produced via genetic engineering procedure, by preparing anucleic acid molecule having a gene sequence encoding the amino acidsequence of the monoclonal antibody protein. A person skilled in the artwould also be familiar with techniques for modifying a monoclonalantibody using genetic information about, e.g., H chains, L chains,variable regions thereof, and CDR sequences thereof to thereby improvethe binding ability and specificity of the antibody, and techniques forpreparing an antibody suitable for a therapeutic agent by altering ananimal antibody such as a mouse antibody into a human-type antibody. Ahuman-type monoclonal antibody can also be prepared by sensitizing anon-human transgenic animal carrying a human antibody gene to anantigen. Another method which does not require sensitization of ananimal is a technique involving: preparing a phage library expressing anantigen binding region of a human antibody or a part thereof (humanantibody phage display); obtaining a phage clone expressing a peptidewhich specifically binds to a corresponding antigen or an antibodyhaving a desired amino acid sequence; and producing a desired humanantibody based on the information of the selected phage clone. A personskilled in the art can employ such a technique as appropriate (see,e.g., a review by Taketo Tanaka et al., Keio J. Med., Vol. 60, pp.37-46). A person skilled in the art can also design an antibody to beadministered to a non-human animal in a similar manner to a humanizedantibody, by using information about amino acid sequences of CDRs andvariable regions as appropriate.

The term “antigen-antibody reaction” used herein means that an antibodybinds to an IGF-I receptor with an affinity represented by anequilibrium dissociation constant (KD) of 1×10⁻⁸M or less. The antibodyof the present invention should preferably bind to an IGF-I receptorwith a KD of usually 1×10⁻⁸M or less, particularly 1×10⁻⁹M or less, moreparticularly 1×10⁻¹⁰M or less.

The term “specificity” of an antibody used herein means that an antibodycauses a strong binding based on antigen-antibody reaction to a specificantigen. In the context of the present invention, the IGF-Ireceptor-specific antibody means an antibody which, when used at aconcentration sufficient to significantly cause antigen-antibodyreaction with cells expressing an IGF-I receptor, causesantigen-antibody reaction with an INSR at a reactivity of 1.5 times orless the reactivity with a Mock cell. An INSR has a high similarity toan IGF-I receptor in primary structure (amino acid sequence) andhigher-order structure.

A person skilled in the art would be able to carry out measurement ofantigen-antibody reaction by selecting an appropriate binding assay in asystem of a solid phase or liquid phase. Examples of such assaysinclude, although not limited to: enzyme-linked immunosorbent assay(ELISA), enzyme immunoassay (EIA), surface plasmon resonance (SPR),fluorescence resonance energy transfer (FRET), and luminescenceresonance energy transfer (LRET). Measurement of antigen-antibodybinding affinity can be carried out by, e.g., labelling an antibodyand/or an antigen with, e.g., an enzyme, a fluorescent material, aluminescent material, or a radioisotope, and detecting theantigen-antibody reaction using a method suitable for measuring thephysical and/or chemical properties characteristic to the label used.

The anti-IGF-I receptor antibodies according to the present inventionencompass both an agonist antibody and an antagonist antibody. When usedsingly, the IGF-I receptor agonist antibody of the present invention hasan effect of enhancing the growth activity of myoblasts. When used incombination with IGF-I, the IGF-I receptor antagonist antibody of thepresent invention has an effect of inhibiting the IGF-I-induced growthactivity of myoblasts.

The IGF-I receptor agonist antibody according to the present inventionwhich binds strongly to a specific domain of the IGF-I receptor has aneffect of enhancing the growth activity of myoblasts in vitro.

The IGF-I receptor agonist antibody of the present invention does nothave an effect of enhancing glucose uptake by differentiated musclecells in vitro at an effective concentration sufficient to enhance thegrowth activity of myoblasts, preferably at a concentration 10 times ashigh as the effective concentration, more preferably at a concentration100 times as high as the effective concentration.

While IGF-I has remarkable hypoglycemic effect at a dosage sufficient toexhibit muscle-mass increasing effect, the IGF-I receptor agonistantibody of the present invention does not have hypoglycemic effect atan effective dosage sufficient to exhibit an effect of increasing musclemass, preferably at a dosage 10 times as high as the effective dosage.

In addition, the IGF-I receptor agonist antibody, when administered to aguinea pig at a single dose, exhibit an in vivo activity to increase themuscle mass effect which corresponds to the activity achieved bysustained administration of IGF-I. The IGF-I receptor agonist antibodyof the present invention also has a long half-life in blood, andexhibits muscle-mass increasing effect via single-dose administration toan animal.

Thus, the IGF-I receptor agonist antibody of the present invention has apotential as a therapeutic or prophylactic agent for a variety ofdiseases associated with the IGF-I receptor such as disuse muscleatrophy and dwarfism, for which IGF-I has also been expected to beeffective. In addition, the IGF-I receptor agonist antibody of thepresent invention can solve the problems involved in IGF-I by, e.g.,overcoming the hypoglycemic effect and extending the blood half-life.

The IGF-I receptor antagonist antibody of the present invention inhibitsthe binding of IGF-I to the IGF-I receptor. According to one embodimentof the IGF-I receptor antagonist antibody of the present invention, theantibody activates the IGF-I receptor while inhibiting the effects ofIGF-I on the IGF-I receptor. In this embodiment, the antibody has aneffect of negating an additive agonistic activity with IGF-I, e.g., aneffect of negating the activity of IGF-I to induce growth of myoblasts.Another embodiment of the IGF-I receptor antagonist antibody of thepresent invention binds to, but does not activate, the IGF-I receptor.Examples of such antagonist antibodies which does not cause activationof the IGF-I receptor via cross-linking include, although not limitedto: antibodies having monovalent antigen-binding ability, such as Faband scFv; and antibodies having divalent binding sites, such asbispecific antibodies, in which only one of the binding sites binds to aspecific domain of the IGF-I receptor, or in which the binding sites arespaced with a controlled interval using a linker. When preparing theIGF-I receptor antagonist antibody according to the present invention,it is possible to confirm whether the antibody binds to the IGF-Ireceptor but lacks agonistic activity by: determining whether theantibody has binding ability to the IGF-I receptor using a method ofmeasuring antigen-antibody reactivity between the antibody and the IGF-Ireceptor; or determining whether the antibody lacks an activity toinduce cell proliferation using a cell proliferation test with, e.g.,myoblasts. On the other hand, the IGF-I receptor antagonist antibodydoes not affect glucose uptake by differentiated muscle cells in vitroor the blood glucose level in vivo. Therefore, the IGF-I receptorantagonist antibody of the present invention has a potential as atherapeutic or prophylactic agent without adverse effects such ashyperglycemia, and can be used for treating malignant tumors such asbreast cancer, bowel cancer, sarcoma, lung cancer, prostate cancer,thyroid cancer, and myeloma.

[Binding Ability of the Anti-IGF-I Receptor Antibody]

The anti-IGF-I receptor antibody according to the present invention binsto the CR domain of the IGF-I receptor as an epitope. On the other hand,the IGF-I receptor agonist antibody does not have an ability to bind toINSR, which has a high similarity to the IGF-I receptor in primarystructure (amino acid sequence) and higher-order structure.

By binding to the CR domain of the IGF-I receptor, the anti-IGF-Ireceptor antibody according to the present invention is deemed toactivate a homo-type receptor, which is a dimer of two copies of theIGF-I receptor, or a hetero-type receptor, which is a dimer between theIGF-I receptor and INSR.

[Sequence of the Anti-IGF-I Receptor Antibody]

The sequence of the anti-IGF-I receptor antibody according to thepresent invention is not particularly limited, as long as itspecifically binds to an IGF-I receptor of a vertebrate and has anactivity to induce cell proliferation.

However, the antibody should preferably have specific amino acidsequences as CDR sequences, as will be explained in details below. Inthe context of the present invention, the term “identity” of amino acidsequences used herein means the ratio of identical amino acid residuesbetween the sequences, while the term “similarity” of amino acidsequences used herein means the ratio of identical or similar amino acidresidues between the sequences. The similarity and identity of aminoacid sequences can be determined, e.g., using BLAST method (with defaultconditions of PBLAST provided by NCBI).

The term “similar amino acid residues” used herein means a group ofamino acid residues having side chains with similar chemical properties(e.g., electric charge or hydrophobicity). Groups of similar amino acidresidues include:

1) amino acid residues having aliphatic side chains: glycine, alanine,valine, leucine, and isoleucine residues;2) amino acid residues having aliphatic hydroxyl side chains: serine andthreonine residues;3) amino acid residues having amide-containing side chains: asparagineand glutamine residues;4) amino acid residues having aromatic side chains: phenylalanine,tyrosine, and tryptophan residues;5) amino acid residues having basic side chains: lysine, arginine, andhistidine residues;6) amino acid residues having acidic side chains: aspartic acid andglutamic acid residues; and7) amino acid residues having sulfur-containing side chains: cysteineand methionine residues.

According to the present invention, the sequence of CDR-1 of the heavychain variable region (CDR-H1) should preferably be the amino acidsequence defined in SEQ ID NO:3 (SerTyrTrpMetHis) or an amino acidsequence derived from SEQ ID NO:3 via substitution, deletion orinsertion of any one amino acid residue. The sequence of CDR-H1 shouldalso preferably have a similarity of 80% or higher to SEQ ID NO:3. Inthe context of the present invention, when an amino acid residue(hereinafter “the first amino acid residue”) of an amino acid sequenceis substituted with another amino acid residue (hereinafter “the secondamino acid residue”), the first amino acid residue before thesubstitution and the second amino acid residue after the substutionshould more preferably be similar to each other in structure and/orcharacteristics.

The sequence of CDR-2 of the heavy chain variable region (CDR-H2) shouldpreferably be the amino acid sequence defined in SEQ ID NO:4(GluThrAsnProSerAsnSerValThrAsnTyrAsnGluLysPheLysSer) or an amino acidsequence derived from SEQ ID NO:4 via substitution, deletion orinsertion of any one or two amino acid residues. The sequence of CDR-H2should also preferably have a similarity of 82% or higher, particularly88% or higher, more particularly 94% or higher to SEQ ID NO:4.

The sequence of CDR-3 of the heavy chain variable region (CDR-H3) shouldpreferably be the amino acid sequence defined in SEQ ID NO:5(GlyArgGlyArgGlyPheAlaTyr) or an amino acid sequence derived from SEQ IDNO:5 via substitution, deletion or insertion of any one or two aminoacid residues. The sequence of CDR-H3 should also preferably have asimilarity of 75% or higher, particularly 87% or higher to SEQ ID NO:5.

The sequence of CDR-1 of the light chain variable region (CDR-L1) shouldpreferably be the amino acid sequence defined in SEQ ID NO:6(ArgAlaSerGlnAsnIleAsnPheTrpLeuSer) or an amino acid sequence derivedfrom SEQ ID NO:6 via substitution, deletion or insertion of any one ortwo amino acid residues. The sequence of CDR-L1 should also preferablyhave a similarity of 81% or higher, particularly 90% or higher to SEQ IDNO:6.

The sequence of CDR-2 of the light chain variable region (CDR-L2) shouldpreferably be the amino acid sequence defined in SEQ ID NO:7(LysAlaSerAsnLeuHisThr) or an amino acid sequence derived from SEQ IDNO:7 via substitution, deletion or insertion of any one amino acidresidue. The sequence of CDR-L2 should also preferably have a similarityof 85% or higher to SEQ ID NO:7.

The sequence of CDR-3 of the light chain variable region (CDR-L3) shouldpreferably be the amino acid sequence defined in SEQ ID NO:8(LeuGlnGlyGlnSerTyrProTyrThr) or an amino acid sequence derived from SEQID NO:8 via substitution, deletion or insertion of any one or two aminoacid residues. The sequence of CDR-L3 should also preferably have asimilarity of 77% or higher, particularly 88% or higher to SEQ ID NO:8.

Still more preferably, the anti-IGF-I receptor antibody according to thepresent invention should have the combination of CDR sequences of: asthe CDR-H1 sequence, the amino acid sequence defined in SEQ ID NO:3; asthe CDR-H2 sequence, the amino acid sequence defined in SEQ ID NO:4; asthe CDR-H3 sequence, the amino acid sequence defined in SEQ ID NO:5; asthe CDR-L1 sequence, the amino acid sequence defined in SEQ ID NO:6; asthe CDR-L2 sequence, the amino acid sequence defined in SEQ ID NO:7; andas the CDR-L3 sequence, the amino acid sequence defined in SEQ ID NO:8.

Methods for identifying the sequence of each of CDR-H1, CDR-H2, CDR-H3,CDR-L1, CDR-L2, and CDR-L3 of an antibody include: Kabat method (Kabatet al., The Journal of Immunology, 1991, Vol. 147, No. 5, pp. 1709-1719)and Chothia method (Al-Lazikani et al., Journal of Molecular Biology,1997, Vol. 273, No. 4, pp. 927-948). These methods are within thetechnical common knowledge to persons skilled in the art, the summariesthereof being available, e.g., on the website of Dr. Andrew C. R.Martin's Group (http://www.bioinf.org.uk/abs/).

The framework sequences of immunoglobulin for the antibody of thepresent invention should preferably be the framework sequences of eachclass of immunoglobulin of a vertebrate, more preferably the frameworksequences of each class of immunoglobulin of a human or a non-humananimal including guinea pig, monkey, rabbit, cow, pig, horse, sheep,dog, fowl, mouse, or rat.

The anti-IGF-I receptor antibody according to the present inventionshould preferably have specific amino acid sequences as the heavy chainvariable region and the light chain variable region, as will bespecified below.

The heavy chain variable region should preferably have the amino acidsequence defined in SEQ ID NO:9, an amino acid sequence derived from SEQID NO:9 via substitution, deletion or insertion of any one or two aminoacid residues, or an amino acid sequence having a similarity of 90% orhigher to SEQ ID NO:9. The light chain variable region should preferablyhave the amino acid sequence defined in SEQ ID NO:10, an amino acidsequence derived from SEQ ID NO: 10 via substitution, deletion orinsertion of any one or two amino acid residues, or an amino acidsequence having a similarity of 90% or higher to SEQ ID NO:10. Theanti-IGF-I receptor antibody according to the present invention shouldmore preferably be IGF11-16, that is, should include the combination ofSEQ ID NO:9 as the heavy chain variable region and SEQ ID NO:10 as thelight chain variable region.

A person skilled in the art will be able to design a humanizedanti-IGF-I receptor antibody according to the present invention byselecting amino acid sequences of CDRs and/or variable regions of aheavy chain and a light chain from those mentioned above and combiningthem with amino acid sequences of framework regions and/or constantregions of a heavy chain and a light chain of a human antibody asappropriate. Amino acid sequences of framework regions and/or constantregions of a heavy chain and a light chain of a humanized antibody canbe selected from, e.g., those of human IgG, IgA, IgM, IgE, and IgDclasses or variants thereof.

When the anti-IGF-I receptor antibody according to the present inventionis an IGF-I receptor agonist antibody, the antibody of the presentinvention or its antigen binding fragment should preferably be human IgGclass or a variant thereof, more preferably human IgG4 subclass, humanIgG1 subclass, or a variant thereof. According to one example, astabilized IgG4 constant region has proline at position 241 in the hingeregion according to Kabat's numbering system. This position correspondsto position 228 in the hinge region according to EU numbering system(Kabat et al., Sequences of Proteins of Immunological Interest, DIANEPublishing, 1992, Edelman et al., Proc. Natl. Acad. Sci USA, 63, 78-85,1969). The residue at this position in human IgG4 is usually serine,while substitution of serine with proline can induce stabilization ofthe resultant antibody. According to another example, introduction ofN297A mutation into the constant region of IgG1 serves to minimize theability to bind to the Fc receptor and/or the ability to fix acomplement.

[Competitive Binding]

A humanized antibody which causes competitive binding to the IGF-Ireceptor with the humanized anti-IGF-I receptor antibody according tothe present invention is also included in the scope of the presentinvention. The term “competitive binding” used herein means thephenomenon that when there are two or more monoclonal antibodiestogether with an antigen, the binding of one of the antibodies to theantigen is inhibited by the binding of the other antibody to theantigen. The competitive binding can usually be measured by, e.g.,adding, to a constant amount (concentration) of a monoclonal antibody,another monoclonal antibody with varying the amount (concentration)thereof, and determining the amount (concentration) of the lattermonoclonal antibody at which the binding amount of the former monoclonalantibody, existing in the constant amount, is decreased. The degree ofinhibition thereof can be expressed in the unit of IC₅₀ or Ki. Themonoclonal antibody which causes competitive binding with the humanizedanti-IGF-I receptor antibody according to the present invention means anantibody having an IC₅₀ of 1000 nM or less, particularly 100 nM or less,more particularly 10 nM or less when measuring an antigen-antibodybinding using the humanized anti-IGF-I receptor antibody according tothe present invention, e.g., the IGF11-16 antibody, at 10 nM.Measurement of competitive binding can also be made by labelling theantibody for use with, e.g., an enzyme, a fluorescent substance, aluminescent substance, a radioactive isotope, etc., and detecting thelabel using a measurement method suitable for detection of the physicaland/or chemical properties of the label.

[Cross-Reaction]

The anti-IGF-I receptor antibody according to the present inventionshould preferably cross-react with the IGF-I receptor of anothervertebrate. The term “cross-reaction” means that while the antibodycauses antigen-antibody reaction with the IGF-I receptor from a targetanimal (such as human), the antibody also has an ability to bind to anantigen derived from another animal different from the target animal.The antibody should preferably has a cross-reactivity with the IGF-Ireceptor of a different animal from the target animal whose IGF-Ireceptor is the target of the antigen-antibody reaction by the antibody,such as human or a non-human animal including guinea pig, monkey,rabbit, cow, pig, horse, sheep, dog, or fowl. Example 7 demonstratesthat an anti-IGF-I receptor antibody, IGF11-16, was shown to bind to theProSerGlyPheIleArgAsnGlySerGlnSerMet (SEQ ID NO: 32) sequence in the CRdomain of the human IGF-I receptor. Since thisProSerGlyPheIleArgAsnGlySerGlnSerMet (SEQ ID NO: 32) sequence isconserved in the homologous parts of the IGF-I receptors of monkey(cynomolgus monkey), rabbit, guinea pig, cow, sheep, horse, and dog,this antibody has cross-binding ability to the IGF-I receptors fromthese species. In addition, since the amino acid sequences of thehomologous parts of mouse and rat are bothProSerGlyPheIleArgAsnSerThrGlnSerMet (SEQ ID NO: 32), screening for ananti-IGF-I receptor antibody which binds to this part makes it possibleto obtain an antibody which binds to the IGF-I receptors of, e.g., mouseand rat, and also has similar characteristics and functions as those ofIGF11-16.

Alternatively, a cell or an animal of a species which does notcross-react with the anti-IGF-I receptor antibody according to thepresent invention can be altered via genetic engineering into a cell oran animal expressing an IGF-I receptor with which the anti-IGF-Ireceptor antibody according to the present invention cross-reacts.

[Activity to Induce Growth of Vertebrate-Derived Cells and Activity toInduce an Increase in the Muscle Mass and/or the Body Length]

An anti-IGF-I receptor antibody according to an embodiment of thepresent invention has an activity to induce growth of vertebrate-derivedcells. Although IGF-I receptor agonist antibodies were already known, noantibody has been reported to show an activity to induce growth primarycultured cells, particularly myoblasts. Also, there has been no knownantibody reported so far as having cell growth-inducing activity at adosage lower than the EC₅₀ value of IGF-I in vitro. Thevertebrate-derived cells in the context of the present invention shouldpreferably be cells derived from mammals, birds, reptiles, amphibia, orfish, more preferably cells derived from mammals or birds, still morepreferably cells derived from human, monkey, rabbit, guinea pig, cow,pig, sheep, horse or dog. Cells derived from these species which expressan IGF-I receptor with which the anti-IGF-I receptor antibody accordingto the present invention cross-reacts can be induced to proliferate bythe anti-IGF-I receptor antibody according to the present invention. Thevertebrate-derived cells according to the present invention alsoencompass: cells and animals engineered to express an IGF-I receptor ofa species with which the anti-IGF-I receptor antibody according to thepresent invention cross-reacts; and modified animal cells derived fromsuch engineered cells and animals.

An antibody's activity to induce growth of vertebrate-derived cells canbe analyzed in vitro using primary cultured cells, established celllines, or transformants derived from such cells. The term “primarycultured cells” means cells which were isolated from an organ or atissue of a living organism, and can typically be subcultured for somepassages. Primary cultured cells derived from a vertebrate can beobtained from an organ or a tissue of the vertebrate via enzymetreatment, dispersion with physical means, or explant method. An organor a tissue or a fragment thereof obtained from the vertebrate can alsobe used for analyzing the antibody's activity above. Preferable examplesof organs and tissues from which primary cells are prepared include:endocrine tissues such as thyroid, parathyroid, and adrenal gland;immune tissues such as appendix, tonsil, lymph nodes, and spleen;respiratory organs such as trachea and lung; digestive organs such asstomach, duodenum, small intestine, and large intestine; urinary organssuch as kidney and urinary bladder; male genital organs such as vasdeferens, testicle, and prostate; female genital organs such as breastand fallopian tube; and muscle tissues such as heart muscle and skeletalmuscles. More preferable examples include liver, kidney, or digestiveorgans or muscle tissues, among which muscle tissues are still morepreferable. Primary cultured cells which can be used for analyzing thegrowth-inducing activity of an anti-IGF-I receptor antibody in thecontext of the present invention are cells which express an IGF-Ireceptor and can be induced to proliferate by IGF-I binding to the IGF-Ireceptor. Typical examples thereof are skeletal muscle myoblasts, whichare primary cultured cells isolated from muscle tissue. Human- oranimal-derived primary cultured cells available by assignment orcommercially on the market can also be obtained and used. Human primarycultured cells are available from various institutions and companies,e.g., ATCC, ECACC, Lonza, GIBCO, Cell Applications, ScienCell researchlaboratories, and PromoCell.

The term “cell line” means a line of cultured cells which were derivedfrom a living organism and then immortalized such that they cansemipermanently proliferate with maintaining their specific properties.Cell lines are divided into non-tumor-derived cell lines andtumor-derived cell lines. Vertebrate-derived cell lines which can beused for analyzing the growth-inducing activity of the anti-IGF-Ireceptor antibody according to the present invention are cells whichexpress an IGF-I receptor and can be induced to proliferate by IGF-Ibinding to the IGF-I receptor. Examples of cell lines which express anIGF-I receptor and can be induced to proliferate by IGF-I include,although not limited to: human neuroblastoma SH-SY5Y, human epidermalkeratinocyte line HaCaT, human alveolar basal epithelial adenocarcinomacell line A549, human colon-adenocarcinoma cell line Caco-2, humanhepatocellular cancer cell line HepG2, human cervical cancer cell lineHela, human cervical cancer cell line SiHa, human breast cancer cellline MCF7, human pluripotent human embryonal carcinoma line NTERA-2, andhuman bone cancer cell line U-2-OS.

Other cells which can be used for analyzing the growth-inducing activityof the anti-IGF-I receptor antibody according to the present inventionare transformants derived from primary cultured cells and cell lines.Examples of such transformants include: iPS cells produced from primarycultured cells; and cells and tissues differentiated from such iPScells. Other transformants include primary cultured cells and cell linesengineered to incorporate a gene so as to transiently or permanentlyexpress the gene. Examples of genes to be introduced into and expressedby such cells include IGF-I receptor genes of human and other species.

Methods for determining the ability of the anti-IGF-I receptor antibodyaccording to the present invention to induce vertebrate-derived cells toproliferate include: cell counting, measurement of DNA synthesis, andmeasurement of change in the metabolic enzyme activity. Methods for cellcounting include methods using blood cell counting plates or cellcounting devices such as Coulter counters. Methods for measuring DNAsynthesis include methods based on uptake of [3H]-thymidine or5-bromo-2′-deoxyulysine (BrdU). Method for measuring the change inmetabolic enzyme activity include MTT method, XTT method, and WSTmethod. A person skilled in the art could also employ other methods asappropriate. An activity to induce cell proliferation is detected if thegrowth of the cultured cells reacted with the anti-IGF-I receptorantibody according to the present invention is higher than the growth ofthe cells in the absence of the antibody. In this case, it is convenientto also measure the induction activity under the same conditions usingIGF-I, which is an original legand for the IGF-I receptor, as a control.

The cultured cells to be tested are reacted with either the anti-IGF-Ireceptor antibody according to the present invention or IGF-I withvarying its concentration, and the concentration at which 50% of themaximum growth activity is exhibited is determined as an EC₅₀ value.When human skeletal muscle myoblasts are used for evaluating the growthactivity, the EC₅₀ value of the anti-IGF-I receptor antibody accordingto the present invention for inducing cell proliferation shouldpreferably be comparable to that of IGF-I, more preferably 1/10 or lessof that of IGF-I, still more preferably 1/20 or less, still further morepreferably 1/50 or less of that of IGF-I. When human skeletal musclemyoblasts are used for evaluating the growth activity, the EC₅₀ value ofthe anti-IGF-I receptor antibody according to the present inventionshould preferably be 0.5 nmol/L or less, more preferably 0.3 nmol/L orless, still more preferably 0.1 nmol/L or less.

Methods for measuring the activity to induce growth ofvertebrate-derived cells in vivo include: a method involvingparenterally administering the anti-IGF-I receptor antibody according tothe present invention to a vertebrate and measuring changes in theweight, size, cell count, etc., for the entire body of the individualwhich received the administration or for an organ or a tissue isolatedfrom the individual; and a method involving using an animal with a graftof vertebrate cells and measuring changes in the weight, size, cellcount, etc., of the graft including vertebrate cells. Measurements forthe entire body of an individual include: measurements of the bodyweight, the body length, and the circumferences of four limbs;measurement of the body composition, using impedance method; andmeasurement of the creatinine height coefficient. Measurements for anorgan, a tissue, or a graft from an individual include: in the case of anon-human animal, a method involving directly recovering the targetorgan, tissue or graft and measuring its weight, size, or the number ofcells included therein. Non-invasive measurements for an organ, atissue, or a graft from an individual include: image analysis usingX-ray photography, CT, and MRI; and contrast methods using tracers withisotopes or fluorescent substances. If the target tissue is skeletalmuscle, then a change in the muscle force can also be used as anindicator. A person skilled in the art could also employ any othermethods as appropriate for analyzing the activity of the anti-IGF-Ireceptor antibody according to the present invention to induce growth ofvertebrate-derived cells in vivo. Methods for measuring the activity ofthe anti-IGF-I receptor antibody according to the present invention toinduce growth of vertebrate-derived cells in vivo include: carrying outmeasurements using, e.g., the methods mentioned above for individualswho received administration of the anti-IGF-I receptor antibodyaccording to the present invention and individuals who receivedadministration of a different antibody other than the anti-IGF-Ireceptor antibody according to the present invention or any othercontrol substance, and comparing the resultant measurements betweenthese individuals.

The anti-IGF-I receptor antibody according to the present invention ischaracterized by having a longer duration of cell-growth inducing effectrelative to the time of contact with the cells compared to the durationof the wild-type IGF-I, and thereby exhibits improved sustainability.According to Example 12, which demonstrates cell proliferation inductionactivities in vitro, when cells were contacted with the wild-type IGF-Iand then washed with culture medium without IGF-I, the cellproliferation induction activity of the wild-type IGF-I disappearedafter the washing. On the other hand, when cells were contacted withIGF11-16 (anti-IGF-I receptor antibody according to the presentinvention) and then washed with culture medium without IGF11-16, thecell growth-inducing activity continued even after the washing.According to Example 16, which compares the kinetics of IGF-I andIGF11-16 (anti-IGF-I receptor antibody according to the presentinvention) in blood, about 50% or higher of the wild-type IGF-Iadministered to an animal disappeared from the blood within 24 hoursafter the administration, while 60% or higher of the IGF11-16 antibodyadministered to an animal remained in the blood even 48 hours after theadministration. Thus, the IGF11-16 antibody was shown to remain in theblood for a long time. These results indicate that the anti-IGF-Ireceptor antibody according to the present invention exhibits along-term effect of inducing cell proliferation both in vitro and invivo.

The anti-IGF-I receptor antibody according to the present invention isalso expected to exhibit an in vivo effect of increasing the muscle massand/or the body length. Specifically, IGF-I has an effect of inducingthe growth and differentiation of myoblasts in skeletal muscles asmentioned above, as well as an effect of broadening muscle fibers. It isexpected that these effects collectively lead to the effect ofincreasing the muscle mass. Like IGF-I, when the anti-IGF-I receptorantibody according to the present invention is administered to ananimal, it also exhibits an effect of increasing the muscle mass of theanimal. The anti-IGF-I receptor antibody according to the presentinvention is the first IGF-I receptor agonist antibody which has beenshown to exhibit an in vivo effect of increasing the muscle mass.

Methods for measuring the activity of the anti-IGF-I receptor antibodyaccording to the present invention to increase the muscle mass include:for the entire body of the individual which received the administration,measurement of the body weight, the body length, and the circumferencesof four limbs; measurement of the body composition, using impedancemethod; and measurement of the creatinine, and height coefficient. Othermethods include: image analysis using X-ray photography, CT, and MRI;contrast methods using tracers with isotopes or fluorescent substances;and measurement of a change in the muscle force. In the case of anon-human animal, a method involving directly recovering the targetorgan, tissue or graft and measuring its weight and/or size can also beused. The effect of increasing the muscle mass can be evaluated by:comparing the muscle mass increases between an individual to which theanti-IGF-I receptor antibody according to the present invention wasadministered and an individual to which the antibody was notadministered; or comparing the muscle masses of an individual before andafter administration of the anti-IGF-I receptor antibody according tothe present invention. The effect of increasing the muscle mass can bedetermined if there is any increase in the muscle mass of an individualbefore and after the administration of the anti-IGF-I receptor antibodyaccording to the present invention. Preferably, the effect achieved byadministration of the anti-IGF-I receptor antibody according to thepresent invention can be determined when there is a difference ofpreferably 103% or higher, more preferably 104% or higher of the musclemass between an individual to which the anti-IGF-I receptor antibodyaccording to the present invention was administered and an individual towhich the antibody was not administered, or of the same individualbetween before and after administration of the anti-IGF-I receptorantibody according to the present invention. IGF-I also plays a role inthe bone growth, and has an effect of increasing the body length (thebody height in the case of the human). Therefore, the anti-IGF-Ireceptor antibody according to the present invention also exhibits aneffect of increasing the body length when administered to an animal. Theeffect of the anti-IGF-I receptor antibody according to the presentinvention in increasing the body length of an individual can bedetermined by measuring the body weight, the body length, and thecircumferences of four limbs of the individual.

[Effects on the Glucose Uptake by Vertebrate-Derived Cells and/or theBlood Glucose Level of an Animal]

An anti-IGF-I receptor antibody according to an embodiment of thepresent invention is characterized by not affecting the intracellularglucose uptake by differentiated muscle cells derived from a vertebrateand/or the blood glucose level of a vertebrate. Specifically, IGF-I isknown to has an effect of increasing the intracellular glucose uptakeand an effect of lowering the blood glucose level as part of its agonisteffects to the IGF-I receptor. On the other hand, although theanti-IGF-I receptor antibody of the present invention functions as anagonist of an IGF-I receptor antibody, it unexpectedly does not inducethe glucose uptake by differentiated muscle cells even at a dosage of100 times or more as high as the in vitro EC₅₀ value for cellgrowth-inducing activity. More particularly, when parenterallyadministered to an animal at a dosage of 10 times or more as high as theeffective dosage sufficient to induce a muscle mass increase, theantibody unexpectedly does not alter the blood glucose level. Inaddition, as properties as an IGF-I receptor antagonist antibody, itscharacteristics of not affecting the intracellular glucose uptake bydifferentiated muscle cells derived from a vertebrate and/or the bloodglucose level of a vertebrate are advantageous effects which serve toavoid the problem of causing hyperglycemia, which problem was desired tobe solved for the antibody to be used for human therapy, but was notsolved by conventional IGF-I receptor antagonist antibodies. Thevertebrate-derived cells according to the present invention shouldpreferably be cells derived from mammals, birds, reptiles, amphibia orfish, more preferably cells derived from mammals or birds, still morepreferably cells derived from human, monkey, rabbit, guinea pig, cow,pig, sheep, horse or dog. The vertebrate-derived cells according to thepresent invention also encompasses: cells and animals engineered toexpress an IGF-I receptor derived from a vertebrate species having across-reactivity with the anti-IGF-I receptor antibody according to thepresent invention; cells and animals engineered to express an IGF-Ireceptor mutated so as to has a binding ability; and cells derived fromsuch engineered animals.

In order to analyze the effect of the anti-IGF-I receptor antibody ofthe present invention in not affecting the intracellular glucose uptakeby vertebrate-derived cells in vitro, it is possible to use primarycultured cells, cell lines, and transformants derived from such cells.The term “primary cultured cells” means cells which were isolated froman organ or a tissue of a living organism, and can typically besubcultured for some passages. Primary cultured cells derived from avertebrate can be obtained from an organ or a tissue of the vertebratevia enzyme treatment, dispersion with physical means, or explant method.Preferable examples of organs and tissues from which primary cells areprepared include: endocrine tissues such as thyroid, parathyroid, andadrenal gland; immune tissues such as appendix, tonsil, lymph nodes, andspleen; respiratory organs such as trachea and lung; digestive organssuch as stomach, duodenum, small intestine, and large intestine; urinaryorgans such as kidney and urinary bladder; male genital organs such asvas deferens, testicle, and prostate; female genital organs such asbreast and fallopian tube; and muscle tissues such as heart muscle andskeletal muscles. More preferable examples include liver, kidney, ordigestive organs or muscle tissues, among which muscle tissues are stillmore preferable.

Primary cultured cells which can be used for analyzing the feature of ananti-IGF-I receptor antibody for not affecting the intracellular glucoseuptake in the context of the present invention are cells which expressan IGF-I receptor and can be induced to cause intracellular glucoseuptake by IGF-I binding to the IGF-I receptor. Typical examples thereofare muscle cells differentiated from skeletal muscle myoblasts, whichare primary cultured cells isolated from muscle tissue.

The “differentiated muscle cells” of the present invention are musclecells which have caused differentiation, and include those which havenot been completely differentiated. For the sake of convenience, theterm “differentiated muscle cells” in the context of the presentinvention refers to cells which have experienced differentiation for atleast about six days from the start of differentiation. Human- oranimal-derived primary cultured cells available by assignment orcommercially on the market can also be obtained and used. Human primarycultured cells are available from various institutions and companies,e.g., ATCC, ECACC, Lonza, GIBCO, Cell Applications, ScienCell researchlaboratories, and PromoCell.

The term “cell line” means a line of cultured cells which were derivedfrom a living organism and then immortalized such that they cansemipermanently proliferate while maintaining their specific properties.Cell lines are divided into non-tumor-derived cell lines andtumor-derived cell lines. Vertebrate-derived cell lines which can beused for analyzing the effect of the anti-IGF-I receptor antibodyaccording to the present invention on the intracellular glucose uptakeare cells which express an IGF-I receptor and can be induced to causeintracellular glucose uptake by IGF-I binding to the IGF-I receptor.Examples of cell lines which express an IGF-I receptor and can beinduced to cause intracellular glucose uptake by IGF-I include, althoughnot limited to: skeletal muscle cells, fat cells, and epidermalkeratinocytes.

Other cells which can be used for analyzing the effect of the anti-IGF-Ireceptor antibody according to the present invention on theintracellular glucose uptake are transformants derived from primarycultured cells and cell lines. Examples of such transformants include:iPS cells produced from primary cultured cells; and cells and tissuesinduced to differentiate from such iPS cells. Other transformantsinclude primary cultured cells and cell lines engineered to incorporatea gene so as to transiently or permanently express the gene. Examples ofgenes to be introduced into and expressed by such cells include IGF-Ireceptor genes of human and other species.

Methods for determining the effect of the anti-IGF-I receptor antibodyaccording to the present invention on the glucose uptake byvertebrate-derived cells include: measurement of the intracellularglucose concentration; measurement of the intracellular uptake of aglucose analog tracer substance; and measurement of a change in theamount of a glucose transporter. Methods for measuring the glucoseconcentration include absorbance measurement methods such as enzymemethod. Methods for measuring the intracellular uptake amount of aglucose analog tracer substance include measurement of the uptake amountof [3H]-2′-deoxyglucose. Methods for measuring a change in the amount ofa glucose transporter include immunocytostaining and western blotting. Aperson skilled in the art could also employ other methods asappropriate. The fact that there is no effect on the intracellularglucose uptake can be confirmed if the intracellular glucose uptake ofthe cultured cells reacted with the anti-IGF-I receptor antibodyaccording to the present invention is almost the same of theintracellular glucose uptake of the cultured cells in the absence of theantibody. In this case, it is convenient to also carry out themeasurement under the same conditions using IGF-I, which is an originallegand for the IGF-I receptor, as a control.

The cultured cells to be tested are treated with either the anti-IGF-Ireceptor antibody according to the present invention or IGF-I withvarying its concentration, and the glucose uptake of the treatment groupis indicated as a percentage when the intracellular glucose uptake ofthe non-treatment group is determined as 100%. When human differentiatedmuscle cells are used for evaluating the glucose uptake, the glucoseuptake achieved by the anti-IGF-I receptor antibody according to thepresent invention should preferably be equal to or less than the glucoseuptake achieved by IGF-I at the same concentration. More preferably, theglucose uptake achieved by the anti-IGF-I receptor antibody according tothe present invention should be 110% or less, still more preferably100%, of the glucose uptake amount of the non-treatment group. Whenhuman differentiated muscle cells are used for evaluating the glucoseuptake, the glucose uptake achieved by the anti-IGF-I receptor antibodyaccording to the present invention added at an amount of 100 nmol/Lshould preferably be 110% or less, more preferably 105% or less, stillmore preferably from 95% to 100%.

Methods for determining the glucose uptake by vertebrate-derived cellsin vivo include: methods involving parenterally administering theanti-IGF-I receptor antibody according to the present invention to avertebrate and determining a change in the glucose content of an organor a tissue of the individual. Methods of measurement for the entirebody of the individual which received the administration include:measurement of the blood glucose level; and hemoglobin A1C usingglycosylated proteins as indicators. Methods of measuring the glucoseuptake for an organ or a tissue of an individual include: in the case ofa non-human animal, directly recovering the target organ or tissue, andcalculating the concentration of glucose or a tracer. Non-invasivemethods for measuring the glucose uptake individual for an organ or atissue of an individual include: image analysis using X-ray photography,CT, and MRI; and contrast methods using tracers with isotopes orfluorescent substances. If the target tissue is a skeletal muscle, thenthe glucose clamp can also be used as an indicator. A person skilled inthe art could also employ any other methods as appropriate for analyzingthe effect of the anti-IGF-I receptor antibody according to the presentinvention on the glucose uptake by vertebrate-derived cells in vivo.

The anti-IGF-I receptor antibody according to the present invention isalso characterized in that when parenterally administered to avertebrate even at an effective dosage sufficient to increase the musclemass of the vertebrate, preferably at a dosage of 10 times or more theeffective dosage, it does not change the blood glucose level of thevertebrate. When evaluating the effect of the anti-IGF-I receptorantibody of the present invention in changing the blood glucose level ofa vertebrate, it is preferred to use an animal belonging to mammals,birds, reptiles, amphibia or fish, more preferably an animal belongingto mammals or birds, still more preferably human, monkey, rabbit, guineapig, cow, pig, sheep, horse or dog. An animal engineered to express anIGF-I receptor of a species which has cross-reactivity with theanti-IGF-I receptor antibody according to the present invention can alsobe used as an animal for evaluating the effect of the anti-IGF-Ireceptor antibody of the present invention in changing the blood glucoselevel. Invasive methods for measuring the blood glucose level includecolorimetric method and electrode method. Examples of enzyme methodsused for detection include glucose oxidase method (GOD method) andglucose dehydrogenase method (GDH method). Non-invasive methods includeoptical measurement methods. A person skilled in the art can also selectany other method as appropriate. In the case of human, the normal rangeof fasting blood glucose level is from 100 mg/dL to 109 mg/dL. Withregard to adverse events in the blood glucose level resulting from adrug administration (Common Terminology Criteria for Adverse Eventsv4.0), the blood glucose level of lower than the range of from 77 mg/dLto 55 mg/dL is defined as an indicative of low blood glucose, while ablood glucose level of higher than the range of from 109 mg/dL to 160mg/dL is defined as an indicative of high blood glucose. A drugadministration is considered as not affecting the blood glucose levelwhen the blood glucose level after the drug administration is higherthan 55 mg/dL and lower than 160 mg/dL, more preferably higher than 77mg/dL and lower than 109 mg/dL. However, the normal value of bloodglucose level and its range of fluctuation vary depending on the animalto which a drug is administered, and even a human subject may not alwayshave a blood glucose level within a normal range at the time of the drugadministration. Accordingly, in the context of the present invention,the anti-IGF-I receptor antibody according to the present inventionshould preferably be considered as not changing the blood glucose levelof a vertebrate to which the antibody is administered when the change inthe blood glucose level of the vertebrate is preferably 30% or less,more preferably 20% or less, still more preferably 10% or less.

[Process for Producing the Anti-IGF-I Receptor Antibody]

The antibody according to the present invention can be produced usingvarious techniques well-known to a person skilled in the art.Specifically, the antibody according to the present invention may be apolyclonal antibody or a monoclonal antibody (Milstein et al., Nature(England), Oct. 6, 1983, Vol. 305, No. 5934, pp. 537-540). A polyclonalantibody according to the present invention can be obtained, forexample, by sensitizing a mammal with a peptide of the IGF-I receptordefined in SEQ ID NO:2 as an antigen, and recovering the resultantantibody from, e.g., the animal's serum. When the peptide is used as anantigen, the peptide may be bound to a carrier protein such as BSA orKLH or coupled with polylysine. Specific examples of peptides which canbe used as an antigen include, although not limited thereto,ProSerGlyPheIleArgAsnGlySerGlnSerMet (SEQ ID NO: 32), a partial sequenceof SEQ ID NO:2. A monoclonal antibody according to the present inventioncan be obtained, for example, by sensitizing a mammal with such anantigen, recovering an immune cell from the mammal, and fusing theimmune cell with a myeloma cell to produce a hybridoma, cloning andculturing the hybridoma, and recovering the resultant antibody from thecultured hybridoma. An example of such a method for obtaining amonoclonal antibody is described in Example 1, and examples ofmonoclonal antibodies obtained thereby include, although not limitedthereto, a monoclonal antibody having the VH amino acid sequence definedin SEQ ID NO:9 and the VL amino acid sequence defined in SEQ ID NO:10(IGF11-16).

Once such a monoclonal antibody is obtained, then a nucleic acidmolecule having a gene sequence encoding the amino acid sequence of theantibody protein, and such a nucleic acid molecule can also be used forproducing the antibody via genetic engineering technique. A personskilled in the art would appreciate various techniques for utilizinggene information about the antibody, such as information of the H chainand the L chain, the variable regions thereof, and the CDR sequences,for modifying the antibody in order to improve its binding ability orspecificity, or altering an animal antibody such as a mouse antibodyinto a human-type antibody, to thereby prepare an antibody having astructure suitable as a therapeutic agent for human. A human-typemonoclonal antibody can also be prepared by using, as an animal to besensitized with an antigen, a non-human transgenic animal into which ahuman antibody gene has been introduced. Another method which does notrequire sensitization of an animal is a technique involving using aphage library expressing an antigen binding region of a human antibodyor a part thereof (human antibody phage display) and obtaining a phageclone expressing a peptide which specifically binds to a correspondingantigen or an antibody having a desired amino acid sequence, andproducing a desired human antibody based on the information of theselected phage clone. A person skilled in the art can employ such atechnique as appropriate (see, e.g., a review by Taketo Tanaka et al.,Keio J. Med., Vol. 60, pp. 37-46).

A method for producing a monoclonal antibody as mentioned above includesculturing a hybridoma which produces the desired antibody and purifyingthe resultant antibody from the culture supernatant via conventionalmeans. Another method for producing a monoclonal antibody as mentionedabove includes providing a hybridoma which produces the desired antibodyor a phage clone obtained from a human antibody phage display, obtaininga gene encoding such an antibody, more specifically, a gene encoding aheavy chain and/or a light chain of immunoglobulin, preparing a vectorexpressing the gene, and introducing the vector into a host cell (mammalcell, insect cell, microorganism, etc.) for production of the antibody.A person skilled in the art could also modify this method by geneticallyengineering the gene encoding a heavy chain and/or a light chain ofimmunoglobulin for introducing a desired trait, and producing ahumanized antibody, an antibody chimeric protein, a low-molecularantibody, or a scaffold antibody using structure information aboutvariable regions or CDR regions of a heavy chain and/or a light chain ofimmunoglobulin, by using known techniques. In order to improve theperformance of the antibody or avoid adverse effects, a person skilledin the art could also introduce an alteration into the structures ofconstant regions or sugar chains of the antibody, by using techniqueswell-known to a person skilled in the art.

The anti-IGF-I receptor antibody according to the present invention canbe obtained using a method well-known to persons skilled in the art.Specifically, while the humanized anti-IGF-I receptor antibody accordingto the present invention is typically a monoclonal antibody (Milstein etal., Nature, 1983, Vol. 305, No. 5934, pp. 537-540), such a monoclonalantibody can be prepared by, e.g., the following method.

This method starts with, for example, preparation of a nucleic acidmolecule encoding the amino acid sequence(s) of a heavy chain and/or alight chain constituting an immunoglobulin of the anti-IGF-I receptorantibody according to the present invention. The nucleic acid moleculemay then be cloned into various vector or plasmids to produce a vectoror plasmid containing the nucleic acid molecule. Next, the nucleic acidmolecule, vector, or plasmid is used to transform a host cell, which maybe selected from, e.g., eukaryotic cells such as mammal cells, insectcells, yeast cells, and plant cells, and bacterium cells. Thetransformed host cell is then cultured under appropriate conditionswhich can allow production of the anti-IGF-I receptor antibody accordingto the present invention. If necessary, the resultant anti-IGF-Ireceptor antibody according to the present invention may be isolatedfrom the host cell. Various methods that can be used for this procedureare well-known to persons skilled in the art.

A method based on immunization of an animal includes preparing anon-human transgenic animal into which a human antibody gene has beenintroduced as the subject animal to be immunized, immunizing the animalusing the IGF-I receptor and/or its partial peptide as an antigen,recovering an immune cell from the animal and fusing it with a myelomacell to form a hybridoma, which is then cloned to produce an antibody,which is then recovered from the culture supernatant using a routinepurification procedure. Example of such a method of obtaining amonoclonal antibody is described in, e.g., WO2013/180238A.

Another available method includes using a phage library expressing avariable region of a desired humanized antibody or a part thereof (humanantibody phage display) to thereby obtain an antibody which specificallybinds to a corresponding antigen or a phage clone having a specificamino acid sequence, whose information is then used for producing thedesired humanized antibody (see, e.g., the review by Taketo Tanaka etal., The Keio Journal of Medicine, Vol. 60, pp. 37-46)

In this connection, a person skilled in the art can produce variousantibodies such as antibody chimeric proteins, low molecule antibodies,and scaffold antibodies using known techniques, e.g., by making agenetic modification to a gene encoding a heavy chain and/or a lightchain of an immunoglobulin for introducing a desired trait, or by usingstructure information of variable regions or CDR regions of a heavychain and/or a light chain of an immunoglobulin. In addition, in orderto improve the performance of the antibody or avoiding side effects, itis possible to introduce a modification into the structure of a constantregion of an antibody or to introduce glycosylation sites of anantibody, using techniques well-known to persons skilled in the art asappropriate.

[Drug Containing the Anti-IGF-I Receptor Antibody]

The anti-IGF-I receptor antibody according to the present invention canbe used as a therapeutic or prophylactic agent for a conditionassociated with IGF-I or a disease caused by any effect on an IGF-Ireceptor. Specifically, conditions associated with IGF-I or diseasesthat can be the target of therapy or prevention using the IGF-I receptoragonist antibody include: disuse muscle atrophy, dwarfism, hepaticcirrhosis, hepatic fibrosis, diabetic nephropathy, chronic renalfailure, Laron syndrome, aging, intrauterine growth restriction (IUGR),cardiovascular protection, diabetes, insulin resistant, metabolicsyndrome, osteoporosis, cystic fibrosis, myotonic dystrophy,AIDS-associated sarcopenia, HIV-associated fat redistribution syndrome,Crohn's disease, Werner's syndrome, X-linked combined immunodeficiencydisease, hearing loss, anorexia nervosa and retinopathy of prematurity,Turner's syndrome, Prader-Willi syndrome, Silver-Russell syndrome,idiopathic short stature, obesity, multiple sclerosis, ulcerous colitis,low muscle mass, myocardial ischemia, and decreased bone density.Diseases that can be the target of therapy or prevention using the IGF-Ireceptor antagonist antibody include: neuroblastoma, striated musclesarcoma, bone cancer, childhood cancer, acromegalia, ovary cancer,pancreas cancer, benignant prostatic hypertrophy, breast cancer,prostate cancer, bone cancer, lung cancer, colorectal cancer, cervixcancer, synovial sarcoma, urinary bladder cancer, stomach cancer, Wilms'tumor, diarrhea associated with metastatic carcinoid and vasoactiveintestinal peptide secreting tumor, vipoma, Verner-Morrison syndrome,Beckwith-Wiedemann syndrome, kidney cancer, renal cell cancer,transitional cell cancer, Ewing's sarcoma, leukemia, acute lymphoblasticleukemia, brain tumor, glioblastoma, non-glioblastomatic brain tumor,meningioma, pituitary adenoma, vestibular schwannoma, primitiveneuroectodermal tumor, medulloblastoma, astrocytoma, oligodendroglioma,ependymoma, choroid plexus papilloma, gigantism, psoriasis,atherosclerosis, vascular smooth muscle restenosis, inappropriatemicrovascular growth, diabetic retinopathy, Graves' disease, systemiclupus erythematosus, chronic thyroiditis, myasthenia gravis, autoimmunethyroiditis, and Behcet's disease. Particularly preferred uses of theanti-IGF-I receptor antibody according to the present invention includeuse as a therapeutic or prophylactic agent of disuse muscle atrophyand/or dwarfism. The anti-IGF-I receptor antibody according to thepresent invention is advantageous in that it does not change the bloodglucose level upon administration.

A drug containing the anti-IGF-I receptor antibody according to thepresent invention may be formulated in the form of a pharmaceuticalcomposition which contains, in addition to the anti-IGF-I receptorantibody according to the present invention, a pharmaceuticallyacceptable carrier and/or any other excipient. Drug formulation using apharmaceutically acceptable carrier and/or any other excipient can becarried out in accordance with, e.g., a method described in theUniversity of the Sciences in Philadelphia, “Remington: The Science andPractice of Pharmacy, 20th EDITION”, Lippincott Williams & Wilkins,2000. Such a therapeutic or prophylactic agent may be provided as aliquid formulation prepared by dissolving, suspending, or emulsifyingthe ingredients into sterile aqueous medium or oily medium, or as alyophilized formulation thereof. A medium or solvent as a diluent forpreparing such a formulation may be an aqueous medium, examples of whichinclude distilled water for injection and physiological saline solution,which may optionally be used with addition of an osmoregulating agent(e.g., D-glucose, D-sorbitol, D-mannitol, and sodium chloride), and/orin combination with a suitable dissolving aid such as an alcohol (e.g.,ethanol), a polyalcohol (e.g., propylene glycol or polyethylene glycol),or a nonionic surfactant (e.g., polysorbate 80 or polyoxyethylenehydrogenated castor oil 50). Such a formulation can also be preparedwith an oily medium or solvent, examples of which include sesame oil andsoybean oil, which can optionally be used in combination with adissolving aid such as benzyl benzoate and benzyl alcohol. Such liquiddrugs may often be prepared using appropriate additives such asbuffering agents (e.g., phosphate buffering agents and acetate bufferingagents), soothing agents (e.g., benzalkonium chloride and procainehydrochloride), stabilizers (e.g., human serum albumin and polyethyleneglycol), preservatives (e.g., ascorbic acid, erythorbic acid, and theirsalts), coloring agents (e.g., copper chlorophyll β-carotene, Red #2 andBlue #1), antiseptic agents (e.g., paraoxybenzoic acid esters, phenol,benzethonium chloride and benzalkonium chloride), thickeners (e.g.,hydroxypropyl cellulose, carboxymethyl cellulose, and their salts),stabilizers (e.g., human serum albumin mannitol and sorbitol), and odorcorrectives (e.g., menthol and citrus aromas). Other alternative formsinclude therapeutic agents or prophylactic agent for application ontomucous membranes, such formulations often containing additives such aspressure-sensitive adhesives, pressure-sensitive enhancers, viscosityregulators, thickening agents and the like (e.g., mucin, agar, gelatin,pectin, carrageenan, sodium alginate, locust bean gum, xanthan gum,tragacanth gum, gum arabic, chitosan, pullulan, waxy starch, sucralfate,cellulose and its derivatives (such as hydroxypropyl methyl cellulose),polyglycerol fatty acid esters, acrylic acid-alkyl (meth)acrylatecopolymers, or their salts and polyglycerol fatty acid esters),primarily for the purpose of imparting mucosal adsorption or retentionproperties. However, the form, solvent and additives for the therapeuticagent or prophylactic agent to be administered to the body are notlimited to these, and appropriately selection may be made by a personskilled in the art.

A drug containing the anti-IGF-I receptor antibody according to thepresent invention may further contain, in addition to the anti-IGF-Ireceptor antibody according to the present invention, another knownagent (active ingredient). A drug containing the anti-IGF-I receptorantibody according to the present invention may be combined with anotherknown agent in the form of a kit. Examples of active ingredients to becombined with the IGF-I receptor agonist antibody include: growthhormone or an analog thereof, insulin or an analog thereof, IGF-II or ananalog thereof, an anti-myostatin antibody, myostatin antagonist,anti-activin type IIB receptor antibody, activin type IIB receptorantagonist, soluble activin type IIB receptor or an analog thereof,ghrelin or an analog thereof, follistatin or an analog thereof, a beta-2agonist, and a selective androgen receptor modulator. Examples of activeingredients to be combined with the IGF-I receptor antagonist antibodyinclude: corticosteroid, antiemetic, ondansetron hydrochloride,granisetron hydrochloride, metoclopramide, domperidone, haloperidol,cyclizine, lorazepam, prochlorperazine, dexamethasone, levomepromazine,tropisetron, cancer vaccine, GM-CSF inhibitor, GM-CSF DNA vaccine,cell-based vaccine, dendritic cell vaccine, recombinant virus vaccine,heat shock protein (HSP) vaccine, homologous tumor vaccine, autologoustumor vaccine, analgesic, ibuprofen, naproxen, choline magnesiumtrisalicylate, oxycodone hydrochloride, anti-angiogenic, antithrombotic,anti-PD-1 antibody, nivolumab, pembrolizumab, anti-PD-L1 antibody,atezolizumab, anti-CTLA4 antibody, ipilimumab, anti-CD20 antibody,rituximab, anti-HER2 antibody, trastuzumab, anti-CCR4 antibody,mogamulizumab, anti-VEGFantibody, bevacizumab, anti-VEGF receptorantibody, soluble VEGF receptor fragment, anti-TWEAK antibody,anti-TWEAK receptor antibody, soluble TWEAK receptor fragment, AMG 706,AMG 386, antiproliferative, farnesyl protein transferase inhibitor,alpha v beta 3 inhibitor, alpha v beta 5 inhibitor, p53 inhibitor, Kitreceptor inhibitor, ret receptor inhibitor, PDGFR inhibitor, growthhormone secretion inhibitor, angiopoietin inhibitor, tumor-infiltratingmacrophage inhibitor, c-fms inhibitor, anti-c-fms antibody, CSF-1inhibitor, anti-CSF-1 antibody, soluble c-fms fragment, pegvisomant,gemcitabine, panitumumab, irinotecan, and SN-38. The dosage of the otheragent used in combination with the anti-IGF-I receptor antibody may bewithin a dosage used for normal therapy, but can be increased ordecreased depending on the situation.

The therapeutic or prophylactic agent according to the present inventioncan be parenterally administered for the purpose of improving symptoms.For parenteral administration, a transnasal agent may be prepared, and aliquid drug, suspension or solid formulation may be selected. Aninjection may be prepared as a different form of parenteraladministration, the injection being selected as subcutaneous injection,intravenous injection, infusion, intramuscular injection,intracerebroventricular injection or intraperitoneal injection. Otherformulations used for parenteral administration include suppositories,sublingual agents, percutaneous agents and transmucosal administrationagents other than transnasal agents. In addition, intravascular localadministration is possible by a mode of addition or coating onto a stentor intravascular obturator.

The dose for an agent for treatment or prevention according to theinvention will differ depending on the patient age, gender, body weightand symptoms, the therapeutic effect, the method of administration, thetreatment time, or the types of active ingredients in the medicalcomposition, but normally it may be administered in the range of 0.1 mgto 1 g and preferably in the range of 0.5 mg to 300 mg of activecompound per administration for adults, once every one to four weeks, oronce every one to two months. Thus, the administration should preferablybe carried out less than once weekly. However, since the administrationdose and frequency will vary depending on a variety of conditions, loweradministration dose and fewer administration frequency than thosementioned above may be sufficient, or administration dose and frequencyexceeding these ranges may be necessary.

[Uses for Non-Human Animals]

An anti-IGF-I receptor antibody according to an embodiment of thepresent invention can be used for livestock or veterinary applicationson non-human animals. Animals being the target of the anti-IGF-Ireceptor antibody according to the present invention for livestock orveterinary applications should preferably be non-human animals belongingto mammals, birds, reptiles, amphibia or fish, more preferably non-humananimals belonging to mammals or birds, still more preferably an animalselected from monkey, rabbit, guinea pig, cow, pig, sheep, horse or dog.Although cow growth hormones and pig growth hormones are currently usedfor increasing milk production of cows and for promoting growth ofpiglets, respectively, these effects are considered to be achieved byIGF-I, whose expression is induced by a growth hormone (see H. Jiang andX. Ge, Journal of Animal Science, Vol. 92, pp 21-29, 2014). Therefore,the agonist effects of the anti-IGF-I receptor antibody according to thepresent invention can be utilized for the purposes of enhancing milkproduction of an animal and promoting growth of a fetus or a new-bornbaby animal. Examples of other applications for which the anti-IGF-Ireceptor antibody according to the present invention can be usedinclude, although not limited to: increasing the muscle mass of ananimal, increasing the weight ratio of muscles to fat of an animal,increasing the transformation efficiency of fed diet into tissues of thebody, increasing the reproductive efficiency, enhancing the reproductionability of an species for preservation thereof, and treating trauma andexhaustive symptoms involved in debilitating diseases. The antagonisteffect achieved by another embodiment of the anti-IGF-I receptorantibody according to the present invention can be utilized for treatingmalignant tumor of an animal, controlling the reproduction frequency ofan animal, controlling the growth of an individual, and other uses. Aperson skilled in the art could also modify the structure of theanti-IGF-I receptor antibody according to the present invention asappropriate to alter the amino acid sequences of the frameworks orconstant regions of the antibody and to thereby decrease itsimmunogenicity, depending on the animal species to which the antibody isadministered.

[Method for Culturing Cells Using the Anti-IGF-I Receptor Antibody]

IGF-I and its derivatives are widely used in cell culture techniques formaintaining, growing, and/or differentiating vertebrate-derived cells invitro, and commercially marketed as cell culture reagents. However,since IGF-I can lose its effects during long-term culturing due to,e.g., its lack of sufficient stability, it is necessary to, e.g., keepadjusting the concentration thereof in order to carry out cell culturingstably. In addition, since IGF-I induces glucose uptake by cells, thereis a possibility that the metabolism and characteristics of the cellsmay be changed due to an increase in the intracellular glucoseconcentration, and that the culture conditions may change due to adecrease in the glucose concentration of the culture medium. Compared toIGF-I, the anti-IGF-I receptor antibody according to the presentinvention is characterized in that it is more stable, can maintain itscell proliferation effect even after contact with cells, can exhibit anactivity to induce cell proliferation even at a lower concentration, anddoes not induce intracellular glucose uptake. The anti-IGF-I receptorantibody according to the present invention can be used for cellculturing, by adding an appropriate amount of the antibody to culturemedium or by adsorbing or immobilizing an appropriate amount of theantibody to a solid phase of a culture vessel. Thus, the anti-IGF-Ireceptor antibody according to the present invention makes it possibleto reduce the amount to be used, and effectively induce proliferation ofcells adhering to the solid phase. The vertebrate-derived cellsaccording to the present invention should preferably be cells derivedfrom mammals, birds, reptiles, amphibia or fish, more preferably cellsderived from mammals or birds, still more preferably cells derived fromhuman, monkey, rabbit, guinea pig, cow, pig, sheep, horse or dog. Thecells used may be primary cultured cells, cell lines, transformantsderived from such cells, or cells derived from a transgenic animal. Morespecifically, examples of subjects that can be cultured using theanti-IGF-I receptor antibody according to the present invention alsoinclude an organ or a tissue of a vertebrate or a transgenic animalderived from such a vertebrate. The anti-IGF-I receptor antibodyaccording to the present invention can be used for culturing cells forthe purposes of cellular production of a substance or cell therapy andregeneration medicine using such cells.

EXAMPLES Example 1: Production of Mouse Monoclonal Antibody

A mouse monoclonal antibody can be produced by a hybridoma techniquedeveloped by Kohler, et al. (Nature 256: 495-497, 1975). An IGF-Ireceptor agonist antibody was produced by immunizing mice with cellsexpressing a human IGF-I receptor according to standard hybridomatechnology. All animal experiments were conducted in accordance with theregulations of the institution. A standard method involving fusion ofmouse spleen-derived cells with a mouse myeloma cell line (P3U1) wasconducted. Hybridomas were selected using a medium containinghypoxanthine, aminopterin, and thymidine. The hybridoma broth was usedfor evaluation of the affinity by Cell ELISA using cells expressing anIGF-I receptor and evaluation of the activation of intracellulartyrosine kinase of the IGF-I receptor by PATHHUNTER to select a positivehybridoma-containing well. The hybridomas contained in this well weresingle-cloned by a limiting dilution technique. This single-clonedpositive hybridoma was serum-free cultured, and the monoclonal antibodywas purified from the broth through a protein A column (Ab-Capcher,ProteNova). An IGF-I receptor agonist antibody, named IGF11-16, wasfound by evaluation of human myoblast proliferation activity using themonoclonal antibody.

Example 2: Determination of Antibody Isotype

In order to determine the antibody isotype of the IGF-I receptor agonistantibody, ELISA was implemented using antibodies specific to respectiveantibody isotypes. An anti-mouse-IgG antibody (TAGO, 6150) diluted2000-fold with PBS was added to a 96-well plate (Nunc, MaxiSorp) in anamount of 50 μL/well and was left to stand at 4° C. overnight. Thesolution in the 96-well plate was replaced with 3% BSA/PBS, and theplate was used in ELISA. The IGF-I receptor agonist antibody was addedto the anti-mouse-IgG antibody-immobilized 96-well plate in an amount of30 μL/well, followed by reaction at room temperature for 1.5 hours. Eachwell was washed with a washing liquid, and antibodies specificallyreacting with respective isotypes of mouse IgG: anti-mouse-IgG1antibody-ALP conjugate (SBA, 1070-04), anti-mouse-IgG2a antibody-ALPconjugate (SBA, 1080-04), anti-mouse-IgG2b antibody-ALP conjugate (SBA,1090-04), and anti-mouse-IgG3 antibody-ALP conjugate (SBA, 1100-04),were then added in an amount of 30 μL/well, followed by reaction at roomtemperature for 1 hour. A substrate (PNPP) was added in an amount of 100μL/well, followed by reaction at room temperature for 45 minutes. Thedifference between absorbance values at 405 and 550 nm was calculatedand was evaluated as avidity.

Since IGF11-16 showed reactivity with the anti-mouse-IgG1 antibody, theisotype of the antibody was IgG1.

Example 3: Determination of Sequence of Antibody

In order to determine the gene sequences of the light chain and heavychain of the IGF-I receptor agonist antibody, a SMARTER RACE method wasimplemented. Gene fragments encoding the heavy chain and the light chainof the antibody and containing initiation and termination codons wereproduced from RNA derived from the hybridoma producing the antibody bythe SMARTER RACE method, and the nucleotide sequences thereof weredetermined. A first strand cDNA was synthesized using the total RNAderived from the hybridoma as a template with SMARTER RACE 5′/3′ Kit(634859, Clontech) and was then amplified by PCR reaction. Using thecDNA as a template, PCR reaction was performed with the primer touniversal sequence primers attached to the kit and specific to the heavychain and the light chain of the antibody, respectively. The primers forthe light chain (kappa) of the mouse antibody and the IgG1 of the mouseantibody were designed with reference to Accession Nos. BC080787 andLT160966, respectively. The designed nucleotide sequence of the primerfor the light chain of the mouse antibody was ggtgaagttgatgtcttgtgagtgg(SEQ ID NO: 33), and the designed nucleotide sequence of the primer forthe heavy chain of the mouse antibody was gctcttctcagtatggtggttgtgc (SEQID NO: 34). These primers were used in experiments. The resulting PCRproducts were used as 5′ RACE PCR products in TA cloning.

In the TA cloning, the 5′ RACE PCR products were subjected toelectrophoresis, and cDNA having the target sequence was purified withQIAEX II Gel Extraction Kit (20021, Qiagen). The purified cDNA wassubjected to reaction using TaKaRa-Taq (R001A, Takara) at 72° C. for 5minutes to attach adenine to the 5′ and 3′ ends. The cDNA was clonedinto Topoisomerase I-activated PCR II-TOPO vector (hereinafter, referredto as TOPO vector) using TOPO TA-CLONING Kit (450641, Thermofisher)according to the protocol attached to the kit. The TOPO vector clonedwith the target cDNA was transformed into E. coli TOP10, followed byculturing in an agar medium containing 50 μg/mL of kanamycin. Theinsertion of the target cDNA into the TOPO vector was verified by colonyPCR. The nucleotide sequence of the cloned cDNA was identified.Similarly, the nucleotide sequence of the 3′ RACE PCR product wasidentified to determine the full-length sequence of the antibody gene.The full-length nucleotide and amino acid sequences of the light chainof IGF11-16 are shown in SEQ ID NO:27 and SEQ ID NO:28, respectively,and the full-length nucleotide and amino acid sequences of the heavychain of IGF11-16 are shown in SEQ ID NO:29 and SEQ ID NO:30,respectively. The amino acid sequences of CDR-H1, CDR-H2, CDR-H3,CDR-L1, CDR-L2, CDR-L3, the heavy chain variable region, and the lightchain variable region of IGF11-16 are shown in SEQ ID NO:3, SEQ ID NO:4,SEQ ID NO:5, SEQ ID NO:6, SEQ ID NO:7, SEQ ID NO:8, SEQ ID NO:9, and SEQID NO:10, respectively.

Example 4: Avidity to IGF-I Receptor (ELISA)

In order to investigate the avidity of the IGF-I receptor agonistantibody to the IGF-I receptor of human (SEQ ID NO: 2, NP_000866),guinea pig (SEQ ID NO: 11, XP_003475316), cynomolgus monkey (SEQ ID NO:12, NP_001248281), rabbit (SEQ ID NO: 13, XP_017193273), rat (SEQ ID NO:14, NP_494694), and mouse (SEQ ID NO: 15, NP_034643), Cell ELISA wasimplemented using cells expressing the respective IGF-I receptors.

The pEF1 expression vector (Thermofisher) containing the IGF-I receptorgene of human (SEQ ID NO: 16), guinea pig (SEQ ID NO: 17), cynomolgusmonkey (SEQ ID NO: 18), rabbit (SEQ ID NO: 19), rat (SEQ ID NO: 20), ormouse (SEQ ID NO: 21) was transfected into P3U1 cells by lipofection.After the lipofection, the P3U1 cells were cultured at least overnightand were added to a 96-well plate (coated with poly-D-lysine) at aconcentration of 0.8×10⁵ cells/well and immobilized with 10% bufferedformalin (MILDFORM 10NM, Wako), followed by blocking with phosphatebuffer containing 3% BSA. The resulting plate was used for ELISA.

In ELISA, 30 μL of an IGF11-16 antibody solution adjusted to 10 nM with0.1% skimmed milk/3% BSA/PBS was added to each well and was subjected toreaction at room temperature for about 1.5 hours. After washing with awashing liquid twice, anti-mouse-IgG antibody-HRP conjugate solutions(30 μL) adjusted to predetermined concentrations with 0.1% skimmedmilk/3% BSA/PBS were added to respective wells and were subjected toreaction at room temperature for about 1 hour. After washing with awashing liquid twice, 50 μL of a substrate (TMB) was added to each wellto start the reaction. After about 20 minutes, 50 μL of 0.5 M sulfuricacid was added to each well. The absorbances at 450 nm and 550 nm weremeasured, and the difference between absorbance values at 450 nm and 550nm was calculated. The avidity was calculated based on the differencebetween the absorbance values at 450 nm and 550 nm for cells (Mockcells, SEQ ID NO: 22) transfected with a vector not containing the IGF-Ireceptor gene as a standard value 1 (Table 1).

TABLE 1 Cynomolgus Type Mouse Rat Guinea pig Rabbit monkey Human Avidity0.9 1.0 5.3 5.4 5.4 5.4

IGF11-16 increased the avidity to the cells expressing the IGF-Ireceptors of human, guinea pig, cynomolgus monkey, and rabbit by morethan 5-fold compared with Mock cells. In contrast, the avidity ofIGF11-16 to the cells expressing the IGF-I receptors of rat and mousewas almost equivalent to that of Mock cells and was not increased. Theseresults demonstrated that IGF11-16 binds to the human, guinea pig,cynomolgus monkey, and rabbit IGF-I receptors but does not bind to therat and mouse IGF-I receptors.

Example 5: Avidity to Insulin Receptor (ELISA)

In order to investigate the avidity of the IGF-I receptor agonistantibody to an insulin receptor, Cell ELISA was implemented using cellsexpressing human insulin receptor.

The pEF1 expression vector (Thermofisher) containing the human insulinreceptor gene was transfected into HEK 293T cells by lipofection. TheHEK 293T cells after the lipofection were added to a 96-well plate(coated with poly-D-lysine) at a concentration of 0.8×10⁵ cells/well(about 180 μL/well) and were immobilized with 10% buffered formalin(MILDFORM 10NM, Wako), followed by blocking with phosphate buffercontaining 3% BSA. The plate was used in ELISA.

In ELISA, antibody solutions (30 μL) adjusted to predeterminedconcentrations with 0.1% skimmed milk/3% BSA/PBS were added torespective wells and were subjected to reaction at room temperature forabout 1 hour. After washing with a washing liquid (tris buffercontaining Tween) twice, anti-mouse-IgG antibody-ALP conjugate solutions(30 μL) adjusted to predetermined concentrations with 0.1% skimmedmilk/3% BSA/PBS were added to respective wells and were subjected toreaction at room temperature for about 1 hour. After washing with awashing liquid twice, 100 μL of a substrate (PNPP) was added to eachwell to start the reaction. After about 30 minutes, the absorbances at405 nm and 550 nm were measured, and the difference between absorbancevalues at 405 and 550 nm was calculated. The avidity was calculatedbased on the value of the difference between absorbance values at 405and 550 nm for cells (Mock cells, SEQ ID NO: 22) transfected with avector not containing the IGF-I receptor gene and the insulin receptorgene as a standard value 1 (Table 2).

TABLE 2 IGF11-16 human IGF-I receptor human insulin receptor 0.5 nM 2.91.1   5 nM 3.7 1.4

In ELISA using immobilized cells expressing the human IGF-I receptor,the difference between absorbance values at 405 and 550 nm in 0.5 nM and5 nM of IGF11-16 was increased to about 3-fold or more compared withMock cells. In contrast, in ELISA using immobilized cells expressinghuman insulin receptor, the difference between absorbance values at 405and 550 nm in 0.5 nM and 5 nM of IGF11-16 was not increased to 1.5-foldor more. These results demonstrated that IGF11-16 more strongly binds tothe IGF-I receptor compared with the insulin receptor.

Example 6: Analysis of Binding Site of IGF-I Receptor (ELISA)

In order to identify the epitope of the IGF-I receptor agonist antibodyagainst the IGF-I receptor, the avidity of the IGF-I receptor agonistantibody to variants prepared by replacing each domain of the IGF-Ireceptor with a domain of the insulin receptor having a structuresimilar to that of the IGF-I receptor was measured.

An extracellular domain of the human IGF-I receptor (NP_000866) wasreplaced with an extracellular domain of insulin receptor, or anextracellular domain of human insulin receptor (NP_000199) was replacedwith an extracellular domain of the IGF-I receptor. The following foursubstitutions were thereby produced.

Substitution 1: hIGFIR[L1-L2]/hINSR, a substitution in which L1 domainto L2 domain of human insulin receptor were replaced with L1 domain toL2 domain of the human IGF-I receptor;Substitution 2: hINSR[L1-L2]/hIGFIR, a substitution in which L1 domainto L2 domain of the human IGF-I receptor were replaced with L1 domain toL2 domain of the human insulin receptor;Substitution 3: hINSR[L1]/hIGFIR, a substitution in which L1 domain ofthe IGF-I receptor was replaced with L1 domain of the human insulinreceptor; andSubstitution 4: hINSR[L2]/hIGFIR, a substitution in which L2 domain ofthe human IGF-I receptor was replaced with L2 domain of the humaninsulin receptor.

The pEF1 expression vectors (Thermofisher) containing the respectivegenes of the above-mentioned four substitutions of the human IGF-Ireceptor were transfected into P3U1 cells by lipofection. The gene ofhIGFIR[L1-L2]/hINSR as Substitution 1 is shown in SEQ ID NO: 23; thegene of hINSR[L1-L2]/hIGFIR as Substitution 2 is shown in SEQ ID NO: 24;the gene of hINSR[L1]/hIGFIR as Substitution 3 is shown in SEQ ID NO:25; and the gene of hINSR[L2]/hIGFIR as Substitution 4 is shown in SEQID NO: 26. After the lipofection, the P3U1 cells were cultured at leastovernight and were added to a 96-well plate (coated with poly-D-lysine)at a concentration of 0.8×10⁵ cells/well and immobilized with 10%buffered formalin (MILDFORM 10NM, Wako), followed by blocking withphosphate buffer containing 3% BSA. The plate was used in ELISA.

In ELISA, 30 μL of an antibody solution adjusted to 10 nM with 0.1%skimmed milk/3% BSA/PBS was added to each well and was subjected toreaction at room temperature for about 1.5 hours. After washing with awashing liquid twice, 30 μL of an anti-mouse-IgG antibody-HRP conjugatesolution adjusted to 5 nM with 0.1% skimmed milk/3% BSA/PBS was added toeach well and was subjected to reaction at room temperature for about 1hour. After washing with a washing liquid twice, 50 μL of a substrate(TMB) was added to each well to start the reaction. After about 20minutes, 50 μL of 0.5 M sulfuric acid was added to each well to stop thereaction. The absorbances at 450 nm and 550 nm were measured, and thedifference between absorbance values at 450 nm and 550 nm wascalculated. The avidity was calculated based on the difference betweenabsorbance values at 450 nm and 550 nm for cells (Mock cells, SEQ ID NO:22) transfected with a vector not containing the gene of eachsubstitution as a standard value 1 (Table 3).

TABLE 3 Substitution IGF11-16 hIGFIR[L1-L2]/hINSR 5.5hINSR[L1-L2]/hIGFIR 1.5 hINSR[L1]/hIGFIR 5.7 hINSR[L2]/hIGFIR 5.6

In ELISA using immobilized cells expressing hIGFIR[L1-L2]/hINSR,hINSR[L1]/hIGFIR, or hINSR[L2]/hIGFIR, the absorbance at 450 to 550 nmin IGF11-16 was increased to 5-fold or more compared with Mock cells. Incontrast, the avidity of IGF11-16 to the cells expressinghINSR[L1-L2]/hIGFIR was weak. These results demonstrated that IGF11-16binds to a CR domain of the IGF-I receptor.

Example 7: Determination of Epitope of IGF11-16

In order to identify in more detail the epitope from the CR domain as anepitope of IGF11-16, the binding sequence was estimated from the speciesdifference in avidity to the IGF-I receptor of IGF11-16. FIG. 1 showsthe amino acid sequences of the CR domain of the IGF-I receptor of therespective species.

IGF11-16 binds to the human, guinea pig, and rabbit IGF-I receptors, butdoes not bind to the mouse and rat IGF-I receptors. Based on theresults, an amino acid sequence common to human, guinea pig, and rabbitbut not common to mouse and rat was estimated as the epitope of IGF11-16from the amino acid sequences of the CR domain of the IGF-I receptor.

In order to determine the amino acid site of the CR domain of the IGF-Ireceptor to which IGF11-16 binds, the avidity of the CR domain to eachamino acid substitution was measured by ELISA.

Cell ELISA was implemented using cells expressing an IGF-I receptor inwhich the amino acid sequence presumed to bind to IGF11-16 was modifiedin the CR domain.

As the amino acid substitutions of CR domain, the three substitutionsshown below were used. In addition, a wild-type human IGF-I receptor anda wild-type rat IGF-I receptor each incorporated into a pEF1 expressionvector (Thermofisher) were used as a positive control and a negativecontrol, respectively. The expression level of each IGF-I receptor wasdetermined using the reactivity of an FLAG M2 antibody to the FLAG tag(AspTyrLysAspAspAspAspLys; (SEQ ID NO: 35) attached to the intracellulardomain of the IGF-I receptor as an index.

Substitution 1 of the CR domain: in the amino acid sequence of the humanIGF-I receptor (NP_000866, SEQ ID NO: 2), aspartic acid at position 245and alanine at position 247 were replaced with asparagine and threonine,respectively.Substitution 2 of the CR domain: in the amino acid sequence of the humanIGF-I receptor (NP_000866, SEQ ID NO: 2), glutamic acid at position 294was replaced with aspartic acid.Substitution 3 of the CR domain: in the amino acid sequence of the humanIGF-I receptor (NP_000866, SEQ ID NO: 2), glycine at position 315 andserine at position 316 were replaced with serine and threonine,respectively.

HEK 293T cells were seeded in a 10-cm dish coated with poly-D-lysine at9×10⁶ cells/well. On the next day, each plasmid DNA was transfected intothe cells by lipofection. On the following day, the HEK 293T cells weredetached with 0.25% trypsin/EDTA and were suspended in a broth. The HEK293T cells were added to a 96-well plate (coated with poly-D-lysine) ata concentration of 0.8×10⁵ cells/well and were incubated at 37° C. underconditions of 5% CO₂ overnight. The medium was removed from the 96-wellplate, and the cells were immobilized with a 10% buffered formalin(MILDFORM 10NM, Wako). The 10% buffered formalin was replaced with ablocking buffer (3% BSA/PBS/sodium azide), and the plate was used inELISA.

In ELISA, 50 μL of a solution of the IGF11-16 antibody or the FLAG M2antibody adjusted to 1 nM with 0.1% skimmed milk/3% BSA/PBS was added toeach well and was subjected to reaction at room temperature for about 1hour. After washing with a washing liquid twice, anti-mouse-IgGantibody-HRP conjugate solutions (50 μL) adjusted to predeterminedconcentrations with 0.1% skimmed milk/3% BSA/PBS were added torespective wells and were subjected to reaction at room temperature forabout 1 hour. After washing with a washing liquid twice, 100 μL of asubstrate (TMB) was added to each well to start the reaction. Afterabout 30 minutes, 100 μL of 0.5 M sulfuric acid was added to each wellto stop the reaction, and the absorbances at 450 nm was measured. Thevalue of the absorbance at 450 nm was evaluated as the avidity.

The results are shown FIG. 2 . It was confirmed that the reactivities ofthe FLAG M2 antibody with the cells expressing the respectivesubstitutions of the CR domain are substantially equivalent to oneanother and that the expression levels of the individual substitutionsof the CR domain are substantially the same. IGF11-16 increased thevalue of absorbance at 450 nm to 2 or more in the wild-type human IGF-Ireceptor in which no modification was introduced into the CR domain andshowed enhancement in the avidity. IGF11-16 increased the value ofabsorbance at 450 nm to 2 or more in Substitutions 1 and 2 of the CRdomain and showed enhancement in the avidity. In contrast, the value ofabsorbance at 450 nm of Substitution 3 of the CR domain was about 1 andwas the same level as the absorbance of the rat IGF-I receptor as anegative control, and no avidity was recognized. These resultsdemonstrated that the amino acids at positions 315 and 316 of the IGF-Ireceptor are important for the avidity of IGF11-16 to the CR domain ofthe IGF-I receptor.

The results suggest that the binding site of IGF11-16 to the human IGF-Ireceptor is near glycine (Gly) at position 315 and serine (Ser) atposition 316. In general, the recognition sequence of an antibody iscomposed of eight amino acid residues (average of six to ten residues)and IGF11-16 has cross-reactivity showing no avidity to the rat IGF-Ireceptor and showing avidity to the rabbit and human IGF-I receptors;hence, the sequence of the binding site of IGF11-16 to the human IGF-Ireceptor was estimated to be ProSerGlyPheIleArgAsnGly Ser GlnSerMet (SEQID NO: 32) (Gly Ser indicates the amino acid sequence at positions 315and 316).

Example 8: Avidity to IGF-I Receptor Determined by Surface PlasmonResonance

The avidity (binding rate and dissociation rate) of an agent to an IGF-Ireceptor was measured by surface plasmon resonance (SPR).

An anti-His monoclonal antibody was immobilized to a sensor chip CM3(GE) with an Amine Coupling Kit (BR-1000-50, GE) and a His Capture Kit(28-9950-56, GE). The immobilization conditions were NHS/EDC: 7 minutes,50 μg/mL anti-His monoclonal antibody: 3 minutes, ethanolamine: 7minutes, and target: >3000 RU. As analytes, the agent was used atpredetermined concentrations. As a ligand, a recombinant human IGF-Ireceptor His tag (305-GR-050, R&D SYSTEMS, hereinafter, referred to asIGF-IR-His) was used. As a negative control, Purified Mouse IgG2a, K,Isotype Ctrl, Clone: MG2a-53 (401502, BioLegend, hereinafter, referredto as ctrl IgG2a) was used.

The sensor chip CM3 immobilized with the anti-His monoclonal antibodywas set to Biacore T200, the reaction temperature was set to 36° C., anda running buffer (HBS-EP+, BR-1006-69, GE) was fed at a flow rate 30μL/min. The amount of the binding ligand was set to about 100 RU, andIGF-IR-His (0.5 to 2×10⁻⁸ mol/L) was added to the sensor chip to becaptured by the anti-His monoclonal antibody. Ctrl IgG2a (10 nmol/L) wasallowed to react for 1 minute, and HBS-EP+ was fed at a flow rate of 30μL/min for at least 10 minutes. The analyte and HBS-EP+ were added toflow cells (1 and 2) and flow cells (3 and 4), respectively.

The reaction conditions were set to a binding time of 600 seconds and adissociation time of 600 seconds. After completion of the reaction,washing was performed with regeneration buffer 1 (0.2% SDS),regeneration buffer 2 (100 mmol/L Tris-HCl (pH 8.5), 1 mol/L NaCl, 15mmol/L MgCl₂), and regeneration buffer 3 (10 mmol/L glycine-HCl (pH1.5)) for 1 minute each at a flow rate of 30 μL/min. The dissociationrate constant (ka, 1/Ms), binding rate constant (kd, 1/s), anddissociation constant (KD, M) were calculated by analysis with a modelof 1:1 binding using Biacore T200 Evaluation software (ver 2.0). Theresults are shown in Table 4.

TABLE 4 Ligand Analyte ka (1/Ms) kd (1/s) KD (M) IGF-I receptor IGF-I5.099 × 10⁶ 0.009083 1.781 × 10⁻⁹ IGF-I receptor IGF11-16 1.051 × 10⁶ <1× 10⁻⁵*     <1 × 10⁻¹¹ *a value lower than the lower limit of themeasurement of apparatus.

The ka value of IGF11-16 against the human IGF-I receptor was aboutone-fifth of that of IGF-I, indicating a low binding rate. In contrast,the kd value of IGF11-16 against the human IGF-I receptor was lower thanthe lower limit of the measurement of the apparatus and was lower than1/1000 of that of IGF-I, indicating a significantly low dissociationrate and bare dissociation of IGF11-16 bound to the IGF-I receptor. TheKD value of IGF11-16 against the human IGF-I receptor was lower than1/50 of that of IGF-I, indicating a high binding strength. It isdemonstrated that the avidity of IGF11-16 to an IGF-I receptor is highcompared with that of IGF-I.

Example 9: Activation Effect on IGF-I Receptor or Insulin ReceptorDetermined by PATHHUNTER

In order to detect the activation effect of an IGF-I receptor agonistantibody on the IGF-I receptor, the activation of a downstream signal ofthe IGF-I receptor was measured with PATHHUNTER IGF1R Functional Assay(DiscoverX).

A cell line was used in which an adapter protein SHC1-Enzyme Acceptor(EA) fusion protein including an IGF-I receptor and an SH2 domainbinding to an intracellular tyrosine kinase of the IGF-I receptor wasforcibly expressed intracellularly. In order to detect the activationeffect of the IGF-I receptor agonist antibody on an insulin receptor,the activation of a downstream signal of the insulin receptor wasmeasured with PATHHUNTER INSR Functional Assay (DiscoverX). Another cellline was used in which an adapter protein PLCG1-EA fusion proteinincluding an insulin receptor and an SH2 domain that binds to anintracellular tyrosine kinase of the insulin receptor was forciblyexpressed intracellularly. In each cell line, a ligand binds to theIGF-I receptor or the insulin receptor, which causes dimerization of thereceptor; phosphorylation of the receptor to recruit the adapter proteinhaving the SH2 domain; formation of a receptor signal transductioncomplex; acceleration of the binding between the spatially adjacenttyrosine kinase and EA; and reconstitution of the activatedβ-galactosidase. The effect of an agent on a receptor tyrosine kinasecan be identified by measuring the level of the chemiluminescent signalof a substrate hydrolyzed by the reconstituted β-galactosidase activity.

Cells expressing the IGF-I receptor or the insulin receptor were seededin a 96-well plate (Black/clear or White/clear) coated withpoly-D-lysine or collagen-I at 90 μL/well (2×10⁴ cells/well or 5×10³cells/well) and were incubated at 37° C. under conditions of 5% CO₂. Onthe next day, agents in predetermined concentrations were added to theplate at 10 μL/well, followed by incubation at 37° C. under conditionsof 5% CO₂. On the following day, 30 μL of the culture supernatant wasadded to 15 μL of a substrate solution, followed by reaction for 60minutes, and the luminescent signal was measured with a luminometer(Tristar, Berthold Japan K.K.). The activation of the IGF-I receptor wascalculated with the activity of a group in which only a solvent wasdefined as 100%. The results are shown in Table 5.

TABLE 5 Concentration (nM) Agent 0.5 5 50 Control antibody 105 109 109(FLAG M2) Insulin 184 1244 4619 IGF-I 208 3537 5248 IGF11-16 234 29002786

The activation of the insulin receptor was calculated with the activityof a group in which only a solvent was defined as 100%. The results areshown in Table 6.

TABLE 6 Concentration (nM) Agent 0.5 5 50 Control antibody 105 104 111(FLAG M2) Insulin 1432 1655 1405 IGF-I 126 158 240 IGF11-16 95 96 93

The activation of the IGF-I receptor by an agent was measured using acell line expressing the IGF-I receptor. In the cell line expressing theIGF-I receptor, IGF-I and IGF11-16 showed the activation effect on theIGF-I receptor compared with a control.

The activation of the insulin receptor by an agent was measured using acell line expressing the insulin receptor. In the cell line expressingthe insulin receptor, the activation effect on the insulin receptor byinsulin was observed. IGF-I concentration-dependently activated theinsulin receptor, and a significant activation effect was observed at 50nM. In contrast, IGF11-16 did not activate the insulin receptor.

It is known that IGF-I shows reactivity with an insulin receptor. It isalso known that the activation of an insulin receptor causes ahypoglycemic effect. It was demonstrated that IGF11-16 specifically actson the IGF-I receptor and does not have the hypoglycemic effect via theinsulin receptor.

Example 10: Cell Proliferation Activity on Human Myoblast

In order to investigate the proliferation activity of the IGF-I receptoragonist antibody on human myoblasts, an agent was added to humanmyoblasts, and the amount of ATP in the cells after 4 days was measured.

Normal human skeletal muscle myoblasts (HSMM, Lonza) were seeded in a96-well plate (coated with collagen type I) at 0.1 mL/well (2×10³cells/well) using an SkBM-2 (Lonza, CC-3246) medium containing 1% BSAand were incubated at 37° C. under conditions of 5% CO₂. On the next dayof the cell seeding, each agent was added to the plate at 25 μL/well andwas incubated at 37° C. under conditions of 5% CO₂ for 4 days. Theamount of intracellular ATP was measured as an index of cellproliferation with CELLTITER-GLO Luminescent Cell Viability Assay(Promega). The supernatant was removed from the 96-well plate subjectedto the incubation for 4 days so that the broth in each well was 50 μL,and the plate was then left to stand at room temperature for at least 30minutes. CELLTITER-GLO reagent was added to the plate at 50 μL/well,followed by reaction for at least 10 minutes. The luminescent signal wasthen measured with a luminometer (Tristar, Berthold Japan K.K.). Theactivity was calculated with the activity of a group containing only asolvent defined as 100%. The results are shown in Table 7.

TABLE 7 Experiment 1 Experiment 2 Concen- Cell proliferation Cellproliferation tration inducing activity inducing activity Agent (nM) (%)(%) Control antibody 0.005  99 — (FLAG M2 Ab) IGF-I 0.005 102 103IGF11-16 0.005 141 130 16-13 0.005 — 102 26-3 0.005 — 108 Solventcontrol 1* — — 104 (containing NaN₃) Control antibody 0.5  98 — (FLAG M2Ab) IGF-I 0.5 133 137 IGF11-16 0.5 146 143 16-13 0.5 — 109 26-3 0.5 —119 Solvent control 2* — — 112 (containing NaN₃) *Solvent controls 1 and2 contain sodium azide in an amount of 0.005 nM and 0.5 nM,respectively, which are the same amounts as those in antibodies 16-13and 26-3.

IGF-I and IGF11-16 enhanced the cell proliferation activity, comparedwith the control antibody (FLAG M2, Sigma-Aldrich).

The proliferation activity of human myoblasts wasconcentration-dependently enhanced in 0.00005, 0.0005, 0.005, 0.05, 0.5,5, 50, and 500 nM IGF11-16. The EC₅₀ values of the myoblastproliferative activity of IGF11-16 and IGF-I were 0.004 nM and 0.61 nM,respectively. The results indicate that the activity of IGF11-16 wasabove 100 times that of IGF-I.

The antibodies 16-13 and 26-3 described in Non-Patent Literature 35 didnot show noticeable cell proliferation activity compared with thesolvent control (containing sodium azide), and the activity was weakcompared with that of IGF11-16.

Example 11: Cell Proliferation Activity in Guinea Pig Myoblast

Guinea pig myoblasts (Cell Applications) were seeded in a 96-well plate(coated with collagen type I) at 0.1 mL/well (4×10³ cells/well) using anSkBM-2 (Lonza, CC-3246) medium containing 1% BSA and were incubated at37° C. under conditions of 5% CO₂. On the next day of the cell seeding,each agent was added to the plate at 25 μL/well and was incubated at 37°C. under conditions of 5% CO₂ for 4 days. The amount of intracellularATP was measured as an index of cell proliferation by CELLTITER-GLOLuminescent Cell Viability Assay (Promega). The supernatant was removedfrom the 96-well plate subjected to the incubation for 4 days so thatthe broth in each well was 50 μL, and the plate was then left to standat room temperature for at least 30 minutes. CELLTITER-GLO reagent wasadded to the plate at 50 μL/well, followed by reaction for at least 10minutes. The luminescent signal was then measured with a luminometer(Tristar, Berthold Japan K.K.).

The proliferation activity of guinea pig myoblasts wasconcentration-dependently enhanced in 0.00005, 0.0005, 0.005, 0.05, 0.5,5, 50, and 500 nM IGF11-16. The EC₅₀ values of the myoblastproliferative activity of IGF11-16 and IGF-I were 0.004 nM and 0.76 nM,respectively. The results indicate that the activity of IGF11-16 wasabove 100 times that of IGF-I.

Example 12: In Vitro Comparison with Persistence of Effect of IGF-I

In order to compare the persistence of the effects of IGF11-16 andIGF-I, the medium was replaced after 18 hours from the addition ofIGF11-16 or IGF-I, and the proliferation activity of human myoblasts wasmeasured under the conditions that IGF11-16 and IGF-I were removed.

Normal human skeletal muscle myoblasts (Human Skeletal Muscle MyoblastCells, HSMM, Lonza) were seeded in a 96-well plate (coated with collagentype I) at 0.1 mL/well (2×10³ cells/well) using an SkBM-2 (Lonza,CC-3246) medium containing 1% BSA and were incubated at 37° C. underconditions of 5% CO₂. On the next day of the cell seeding, IGF11-16 orIGF-I was added to the plate at 25 μL/well. After 18 hours from theaddition, the medium was replaced with a medium not containing IGF11-16or IGF-I or a medium containing them, followed by incubation at 37° C.under conditions of 5% CO₂ for 4 days. The amount of intracellular ATPwas measured as an index of cell proliferation by CELLTITER-GLOLuminescent Cell Viability Assay (Promega). The supernatant was removedfrom the 96-well plate subjected to the incubation for 4 days so thatthe broth in each well was 50 μL, and the plate was then left to standat room temperature for at least 30 minutes. CELLTITER-GLO reagent wasadded to the plate at 50 μL/well, followed by reaction for at least 10minutes. The luminescent signal was then measured with a luminometer(Tristar, Berthold Japan K.K.). The proportion (control group: 0%)relative to a control group containing only a solvent was calculated asthe cell proliferation activity. The results are shown in FIG. 3 .

The cell proliferation activity increased to 39% and 75%, respectively,in the groups in which 1 nM and 5 nM IGF-I were respectively added for 4days. The cell proliferation activity increased to 8% and 10%,respectively, in the groups in which 1 nM and 5 nM IGF-I wererespectively added for 18 hours and were then washed out, and theactivity was lower than ⅕ of those of the groups in which IGF-I wasadded for 4 days, indicating a noticeable reduction in the effect.

In the group in which 0.5 nM IGF11-16 was added for 4 days, the cellproliferation activity increased to 49%. The cell proliferation activityof the group in which 0.5 nM IGF11-16 was added for 18 hours and wasthen washed out increased to 30%, which corresponded to 60% or more ofthe activity of the group in which IGF11-16 was added for 4 days.

The cell proliferation activities of the group treated with 0.5 nMIGF11-16 in which washing out was performed after addition of an agentwere compared with the cell proliferation activities of the groupstreated with 1 nM and 5 nM IGF-I. The activity of IGF11-16 was high instatistical significance. These results demonstrated that IGF11-16maintains the proliferation activity of human myoblasts even afterwashing out of the agent and has a strong effect compared with IGF-I.IGF11-16 maintained the cell proliferation activity even after washingout. The results indicate that unlike the effect of IGF-I, IGF11-16strongly binds to the IGF-I receptor and has a persistent activationeffect on the IGF-I receptor.

Example 13: Glucose Uptake in Human Differentiated Muscle Cell

In order to investigate the effect of IGF11-16 on the glucose uptake,the uptake amount of radiolabeled ³H-2-deoxy glucose was measured usinghuman differentiated muscle cells and was compared with the effect ofIGF-I.

Normal human skeletal muscle myoblasts (Human Skeletal Muscle MyoblastCells, HSMM, Lonza) were seeded in a 24-well plate (Costar, 3526) at 0.5mL/well (2×10⁴ cells/well) and were incubated at 37° C. under conditionsof 5% CO₂. The medium (an SkBM-2 (Lonza, CC-3246) supplemented with FBS(Lonza, CC-4423W), L-Glutamine (Lonza, CC-4422W), Dexamethasone (Lonza,CC-4421W), rhEGF (Lonza, CC-4420W), and GA-1000 (Lonza, CC-4419W)) wasreplaced with fresh one until the cells were confluent. The medium wasreplaced with 0.5 mL/well of a medium for differentiation (DMEM/F12(1:1) (Gibco, 11320) containing 2% horse serum (Sigma, H1270), 50 U/mLpenicillin, 50 μg/mL streptomycin (Gibco, 15070-063)), and the confluentHSMM cells were incubated at 37° C. under conditions of 5% CO₂ to startdifferentiation into muscle cells. The cells after about 6 days from thestart of differentiation were used as human differentiated muscle cellsin a glucose uptake experiment.

The medium was replaced with 0.5 mL/well of a starvation medium (1 g/Lglucose-containing DMEM (Gibco, 11885) supplemented with 0.1% BSA FattyAcid free (Seikagaku Corporation, 82-002-5), 50 U/mL penicillin, and 50μg/mL streptomycin (Gibco, 15070-063)), and the human differentiatedmuscle cells were incubated at 37° C. under conditions of 5% CO₂overnight. On the next day, the medium was replaced with 0.5 mL/well ofa medium for starvation, and the cells were incubated at 37° C. underconditions of 5% CO₂ for 2 hours. The wells were washed with 1 mL/wellof PBS, and 0.5 mL/well of a treatment medium containing the respectiveagents was added to the wells, followed by incubation at 37° C. underconditions of 5% CO₂ for 2 hours. The treatment medium was prepared togive final concentrations of 0.1 mmol/L glucose, 0.1% BSA, ³H-2-deoxyglucose (1 μCi/mL), and each concentration of a human recombinant IGF-Ior IGF-I receptor agonist antibody using a glucose uptake buffer(containing 20 mmol/L HEPES (DOJINDO, 342-01375), 150 mmol/L NaCl(SIGMA, S5150), 5 mmol/L KCl (Wako, 163-03545), 5 mmol/L MgSO₄ (Wako,131-00405), 1.2 mmol/L KH₂PO₄ (Wako, 169-04245), 25 mmol/L CaCl₂)(Fluka, 21114), and 2 mmol/L pyruvate (Wako, 190-14881) dissolved inwater for injection and having a pH 7.4 adjusted with NaOH). The wellswere washed by adding 1 mL/well of cooled PBS three time to stop theglucose uptake. The cells were lysed by adding 0.25 mL/well of 1 N NaOH.The whole amount of the cell lysate was added to a vial containing 3 mLof liquid scintillator ULTIMA GOLD (PerkinElmer Japan) and was stirred.The radioactivity (DPM) of ³H was measured for 3 minutes with a liquidscintillation counter. The glucose uptake ratio of a treated group wascalculated with the averaged glucose uptake amount (DPM) of an untreatedgroup (control group) defined as 100%. The results are shown in FIG. 4 .

In 0.8, 4, 20, and 100 nM IGF-I, the glucose uptake wasconcentration-dependently and significantly enhanced. In contrast,IGF11-16 did not show significant effect until 100 nM. These resultssuggest that the glucose uptake effect of IGF11-16 in humandifferentiated muscle cells is extremely weak.

Example 14: In Vivo Efficacy (Effect of Increasing Muscle Mass in GuineaPig)

In order verify the in vivo efficacy of the IGF-I receptor agonistantibody, IGF11-16 was administered to guinea pigs in a singleadministration, and the muscle mass after 2 weeks was measured forcomparison with the effect when IGF-I was continuously administered. Theeffect of increasing muscle mass is defined as an effect of increasingthe weight of muscle of a guinea pig by 5% or more compared with that ofthe control group.

IGF11-16 (0.03, 0.1, or 0.3 mg/kg) was subcutaneously or intravenouslyadministered to normal guinea pigs in a single administration. Humanrecombinant IGF-I (Mecasermin) as a positive control was subcutaneouslyembedded with an osmotic pump (Alzet) and was continuously administeredat 1 mg/kg/day. After two weeks from the administration of the agent,the guinea pigs were euthanized by exsanguination under anesthesia, andthe weight of the extensor digitorum longus muscle was measured. Theresults are shown in FIG. 5 .

In the group (iv) of intravenous administration of IGF11-16 in an amountof 0.03, 0.1, or 0.3 mg/kg, the muscle mass was dose-dependently andsignificantly increased, compared with the control group treated withonly a solvent. Even in the group (sc) of subcutaneous administration ofIGF11-16 at 0.3 mg/kg, the muscle mass was significantly increasedcompared with the control group.

The increased amounts of the muscle in the groups of administration of0.03 to 0.3 mg/kg of IGF11-16 in a single administration were equivalentto that in the group (infusion) of continuous administration of 1mg/kg/day of human recombinant IGF-I. The results indicate that IGF11-16shows efficacy even in in vivo by intravenous or subcutaneousadministration in a single administration.

It was demonstrated that IGF11-16 shows efficacy equivalent to that bycontinuous administration of IGF-I in a single administration. Inclinical use, IGF-I (Mecasermin) is administered once or twice a day. Incontrast, IGF11-16 administered one every other week shows in vivoeffectiveness equivalent to that in continuous administration of IGF-I,indicating that IGF11-16 has excellent persistence compared with IGF-I.

Example 15: In Vivo Hypoglycemic Effect (Hypoglycemic Effect in GuineaPig)

In order to verify whether the IGF-I receptor agonist antibody has invivo hypoglycemic effect or not, IGF11-16 was administered to guineapigs in a single administration, and the blood glucose levels weremeasured over time and compared with the hypoglycemic effect of IGF-I ina single administration. The hypoglycemic effect is defined as an effectof lowering the blood glucose level to 50 mg/dL or less or causinghypoglycemia.

IGF-I was subcutaneously administered to guinea pigs a single time, andthe hypoglycemic effect was investigated. The guinea pigs were fastedfor 12 hours, and human recombinant IGF-I (Mecasermin) wassubcutaneously administered to the guinea pigs at 0.3, 1, 3, and 10mg/kg a single time. The guinea pigs were fasted for 24 hours after theadministration. Blood was collected from the awake guinea pigs at beforethe administration (0 hour) and at 1, 2, 4, 8, 10, and 24 hours afterthe administration and was subjected to measurement of the blood glucoselevel with a Glutest Sensor (Sanwa Kagaku Kenkyusyo). The results areshown in FIG. 6 .

IGF-I showed a significant glucose lowering effect at 0.3 mg/kg or more.Hypoglycemia was observed at 1 mg/kg or more. Death was caused at 3mg/kg or more.

IGF11-16 was subcutaneously administered to guinea pigs a single time,and the hypoglycemic effect was investigated. The guinea pigs werefasted for 12 hours, and IGF11-16 was subcutaneously administered to theguinea pigs at 10, 30, and 100 mg/kg a single time. The guinea pigs werefasted for 24 hours after the administration. Blood was collected fromthe awake guinea pigs at before the administration (0 hour) and at 2, 4,8, 10, and 24 hours after the administration and was subjected tomeasurement of the blood glucose level with a Glutest Sensor (SanwaKagaku Kenkyusyo). The results are shown in FIG. 7 .

IGF11-16 did not show any significant difference in the blood glucoselevel, even in the group of 100 mg/kg administration, compared with acontrol group in which only the solvent was administered. The resultsindicate that subcutaneous administration of IGF11-16 does not have ahypoglycemic effect and does not affect the blood glucose level.

IGF11-16 was intravenously administered to guinea pigs a single time,and the hypoglycemic effect was investigated. The guinea pigs werefasted for 12 hours, and IGF11-16 was intravenously administered to theguinea pigs at 0.1, 1.5, 6, and 20 mg/kg. The guinea pigs were fastedfor 24 hours after the administration. Blood was collected from theawake guinea pigs at before the administration (0 hour) and at 0.5, 1,2, 4, 8, and 24 hours after the administration and was subjected tomeasurement of the blood glucose level with a Glutest Sensor (SanwaKagaku Kenkyusyo). The results are shown in FIG. 8 .

IGF11-16 did not show any significant difference in the blood glucoselevel, even in the group of 20 mg/kg administration, compared with acontrol group in which only the solvent was administered. The resultsindicate that intravenous administration of IGF11-16 also does not havea hypoglycemic effect and does not affect the blood glucose level.

IGF11-16 does not have a noticeable hypoglycemic effect in bothsubcutaneous and intravenous administrations, unlike IGF-I, and does notaffect the blood glucose level, indicating that IGF11-16 has apossibility as an agent that overcomes a side effect of IGF-I,hypoglycemia.

Example 16: In Vivo Efficacy (Growth Promoting Effect in Guinea Pig)

In order to verify the in vivo efficacy of the IGF-I receptor agonistantibody on bone, the effect was compared with IGF-I continuouslyadministered and growth hormone (GH) repeatedly administered once a day.IGF11-16 was administered to guinea pigs with removed pituitary gland asingle time. After two weeks, the length of the tibia and the thicknessof the growth plate cartilage were measured as indices of the growthpromoting effect. IGF11-16 (0.3 mg/kg and 1 mg/kg) was subcutaneouslyadministered to the guinea pigs with removed pituitary gland a singletime. As a control, human recombinant IGF-I (Mecasermin) wassubcutaneously embedded with an osmotic pump (Alzet) and wascontinuously administered at 1 mg/kg/day. Another control, humanrecombinant GH (GENOTROPIN), was subcutaneously administered at a doseof 1 mg/kg repeatedly once a day. After two weeks from the agentadministration, the guinea pigs were euthanized by exsanguination underanesthesia, and the thickness of the growth plate cartilage of the tibiaproximal and the length of the tibia were measured. The results areshown in FIGS. 9 and 10 .

In the group (IGF11-16) in which IGF11-16 was subcutaneouslyadministered at 0.3 mg/kg and 1 mg/kg, the thickness of the growth platecartilage and the length of the tibia were dose-dependently andsignificantly extended to show a growth promoting effect, compared withthose in a control group (vehicle) in which the guinea pigs with removedpituitary gland were treated with only the solvent.

The growth promoting effect of the group of a single administration ofIGF11-16 at 0.3 mg/kg was equivalent to that of the group (IGF-I) ofcontinuous administration of human recombinant IGF-I at 1 mg/kg/day. Thegrowth promoting effect of the group of single administration ofIGF11-16 at 1 mg/kg was equivalent to that of the group (GH) of repeatedadministration of human recombinant GH at 1 mg/kg/day. The resultsindicate that a single administration of IGF11-16 shows efficacyequivalent to that of a continuous administration of IGF-I and that ofrepeated administration of GH once a day. In clinical use, humanrecombinant IGF-I (Mecasermin) and human recombinant GH (GENOTROPIN) areadministered by subcutaneous injection once or twice a day and six orseven times a week, respectively. In contrast, IGF11-16 administeredonce every other week shows in vivo effectiveness equivalent to that incontinuous administration of IGF-I and that in repeated administrationof GH once a day, indicating that IGF11-16 has excellent persistencecompared with IGF-I and GH.

Example 17: Kinetics of IGF-I and IGF11-16 in Blood Kinetics of IGF-I inBlood

Guinea pigs were fasted for 12 hours, and human recombinant IGF-I wassubcutaneously administered to the guinea pigs at 0.3, 1, 3, and 10mg/kg. The guinea pigs were fasted for 24 hours after theadministration. Blood was collected from the awake guinea pigs at beforethe administration (0 hour) and at 1, 2, 4, 8, 10, and 24 hours afterthe administration. The human IGF-I concentration in plasma was measuredby ELISA (DG100, R&D). The results are shown in FIG. 11 .

The plasma IGF-I concentration increased administration-dose dependentlyand, after 24 hours from the administration, decreased to about 50% ofthe maximum plasma IGF-I concentration. In the group of 0.3 mg/kgadministration, the IGF-I concentration at 24 hours after theadministration was lower than the lower limit of the measurement. In thegroup of 10 mg/kg administration, the guinea pigs died due tohypoglycemia after 4 hours from the administration, and the plasma couldnot be collected.

Kinetics of IGF11-16 in Blood

Guinea pigs were fasted for 12 hours, and the IGF-I receptor agonistantibody was subcutaneously administered to the guinea pigs at 0.3, 1,3, 10, 30, and 100 mg/kg. The guinea pigs were fasted for 24 hours afterthe administration and were then refed. Blood was collected from theawake guinea pigs at before the administration (0 hour) and at 2, 4, 8,10, 24, 48, and 72 hours after the administration. The IGF11-16concentration in plasma was measured by ELISA. The results are shown inFIG. 12 .

The plasma IGF11-16 concentration increased administration-dosedependently, and the plasma IGF11-16 concentration after 48 hours fromthe administration was retained to be at about 50% or more of that at 24hours after the administration, indicating that the kinetics of IGF11-16in blood is excellent in the persistence compared with that of IGF-I.

INDUSTRIAL APPLICABILITY

The present invention can provide an antibody which specifically bindsto an IGF-I receptor of a vertebrate, and thereby increase the musclemass or the thickness of growth plate cartilage via the IGF-I receptor,but does not reduce the blood glucose level. Therefore, the presentinvention can be used for the treatment, prevention, or diagnosis ofdiseases associated with an anti-IGF-I receptor antibody.

1. An animal with genetic modification for evaluating the activity of ananti-IGF-I receptor antibody or its fragment or a derivative thereof onvertebrate, wherein the animal's innate IGF-I receptor gene which hasbeen mutated in a CR domain thereof and the mutated CR domain encodes anamino acid sequence represented by ProSerGlyPhelleArgAsnGlySerGlnSerMet.2. An animal with genetic modification for evaluating the activity of ananti-IGF-I receptor antibody or its fragment or a derivative thereof onvertebrate, wherein the animal's innate IGF-I receptor gene which hasbeen mutated in a CR domain thereof, and amino acid residue(s) X₁ and/orX₂ of a sequence represented by ProSerGlyPheIleArgAsnX₁×₂GlnSerMet inthe mutated CR domain differs from that of the animal's innate IGF-Ireceptor gene.